Heterologous protein comprising avian alpha-subunit inhibin protein and methods of producing same

ABSTRACT

The present invention relates, in general, to a method of enhancing the production performance of avians, by administering to a bird a heterologous protein comprised of inhibin protein, or a fragment thereof, and a carrier protein. The present invention also relates to a method of enhancing the production performance of avians, by administering to a bird a fusion gene product comprising a gene encoded for the expression of alpha-subunit avian inhibin protein, or a fragment thereof, and a gene encoded for the expression of a carrier protein. An effective amount of the heterologous protein or fusion gene product is administered to an animal such that an immunological response occurs in the animal against the heterologous protein. The present invention further relates to the above heterologous protein and fusion gene product, and to methods of producing the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application of U.S. patent applicationSer. No. 08/395,554 filed Feb. 28, 1995, which is herein incorporated byreference, which is a continuation-in-part of U.S. patent applicationSer. No. 08/202,964, filed Feb. 28, 1994, which is herein incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates, in general, to a method of enhancing theproduction performance of avians, by administering to a bird aheterologous protein comprised of inhibin protein, or a fragmentthereof, and a carrier protein. The present invention also relates to amethod of enhancing the production performance of avians, byadministering to a bird a fusion gene product comprising a gene encodingfor the expression of alpha-subunit avian inhibin protein, or a fragmentthereof, and a gene encoding for the expression of a carrier protein.The present invention further relates to the above heterologous proteinand fusion gene product, and methods of producing the same.

BACKGROUND OF THE INVENTION

Ratites are flightless, generally large, running birds, comprisingseveral orders including the species Ostrich, Emu, Rhea, Cassowaries,and Kiwis. An emu (Dromiceius novaehollandiae) is an Australian ratitebird which is characterized by rudimentary wings and a feathered headand neck. An average adult emu is approximately 6 feet tall and weighsapproximately 150 pounds. An ostrich (Struthio camelius) is a largerunning bird with small wings and thick powerful legs. A standard adultostrich is approximately 8 feet tall and weighs approximately 325 to 375pounds. The term "rhea" is the common name for members of the avianorder Rheiformes. Rheiformes are an order of South American runningbirds, called American ostriches, which differ from the true ostrich intheir smaller size, feathered head and neck, and three-toed feet amongother features.

The ostrich and emu have long had a commercial value in their naturalenvironments of South Africa and Australia, respectively. Ostrichproducts have been in demand for more than 100 years, and a substantialworldwide market exists for their hides, meat, and feathers. Forexample, ostrich leather is used in boots, handbags, jackets, attachecases, wallets, and many other articles. Ostrich feathers are used infashion, costuming, and in feather dusters.

In contrast, the emu is a relative newcomer to the market place. It isvalued for the same products with the addition of an essential oil thatis used in the cosmetic industry. Emu oil, rendered from a thick layerof subcutaneous fat has deep penetrating properties which make the oiluseful in cosmetic creams, such as, wrinkle retardant emollients. Also,possible medicinal uses for emu oil, such as the treatment of arthritis,are currently being investigated. A typical full-grown emu can achieve aheight of 1.6 to 1.9 m or more, and weight 30 to 45 kg or more. Emusmature at about one year, and pre- and post-pubescent emus never showgender specific phenotypic differences. Similar to the ostrich, the emupopulation in the United States has also experienced explosive growthwithin the last several years. As of 1994, there were approximately150,000 total emu, including 15,000 breeding pairs, in the UnitedStates. It is predicted that the numbers of emus in 1995 will furtherincrease to between 500,000 and 750,000 birds, of which 45,000 areexpected to be breeding pairs.

There is a growing demand for ratite products in several countries,including Australia, Belgium, Israel, Canada, the Netherlands, Namibia,South Africa, and Zimbabwe. Accordingly, over the past several yearsthere has been explosive growth in the domestic market for ostrich andemu, and to a lesser extent rhea. In the last five years, the number ofbreeding ostrich pairs and total bird numbers in the United States hasincreased 7.5- and 20-fold, respectively. It is estimated that in 1995,200,000 ostriches, including 20,000 breeding pairs, will exist in theUnited States. The tremendous interest in breeding these animals is dueto the significant value of adults, as well as immature animals, andespecially for proven breeding pairs of ostrich which are valued as highas $75,000.00, with an emu pair valued at $30,000.00 or more. Immatureostriches at three to four months of age are valued at approximately$7,500.00, and immature emu are valued at approximately $5,000.00. Amajority of the animals are purchased between three and six months ofage.

Further, there is tremendous interest in ratites as an alternative tomore traditional forms of animal agriculture. Several factors relatingto ratites make them a superior alternative to the more traditionalforms of animal agriculture (i.e., beef cattle, swine and sheepfarming). These factors include: superior feed conversion ratios, agreater ability to be intensively farmed, large animal size, enhancedreproductive capacity, and exceptional nutritional value of their meat.

For example, ostrich meat, which is a red meat resembling beef, containssignificantly less fat, calories, and cholesterol than chicken or turkeymeat. More particularly, an 85 gram portion of Ostrich meat contains 2grams of fat, 58 mg of cholesterol, and 97 calories. In contrast, an 85gram portion of turkey meat contains 3 grams of fat, 59 mg ofcholesterol, and 135 calories. An 86 gram portion of chicken meatcontains 3 grams of fat, 73 mg of cholesterol, and 140 calories. An 85gram portion of beef (steak) contains 15 grams of fat, 77 mg ofcholesterol, and 240 calories. And finally, an 85 gram portion of porkcontains 19 grams of fat, 84 mg of cholesterol, and 275 calories.(Values for ostrich meat were derived from AMSI Quality LaboratoryReport #0800100. Values for the other meats were derived from U.S.D.A.Handbook No. 8, "Nutritive Value of Foods".) Similar to the ostrich, emumeat is a low fat red meat. More particularly, a 100 gram portion of emumeat contains 1.7 g fat, 57.5 mg cholesterol, and 109 calories. (Valuesfor emu meat were derived from Silliker Laboratories of Texas, Inc.)

Also, ratites such as ostriches provide approximately 100 pounds of meatat the age of 12 months and therefore produce a substantial amount ofmeat in a relatively short period of time.

An illustration of how ratites are a superior alternative to the moretraditional forms of animal agriculture is the following comparison ofan ostrich and a cow. First, an ostrich has a gestation or incubationperiod of 42 days, wherein a cow requires 280 days. Second, an averageostrich produces more than 20 offspring per year, whereas a cow producesone offspring per year. Third, the feed conversion ratio of an ostrichis less than 2:1 whereas the feed conversion ratio of a cow is 5:1.Fourth, the days from conception to slaughter is approximately 407 daysfor an ostrich in contrast to 645 for a cow. Finally, ostriches producefeathers in addition to their meat and leather, whereas cows do notproduce products other than meat and leather.

Considering these attributes, and the increasing needs of the worldpopulation for meat which is nutritious yet low in fat and cholesteroland which may be efficiently produced with a minimum negative impact onthe environment, the ratite industry has a high potential for futuregrowth.

Currently, the demand for ratites far outweighs the supply. However,ratite producers are limited by sub-optimal egg production in mostbreeding age females. Depending on the species, most ratites incaptivity currently lay an average of 10-20 eggs per year whereas theirgenetic potential for egg production is thought to exceed over 60 eggsper year. For example, a high-producing ostrich in the wild can lay anegg approximately every 48 hours during breeding season, and a highproducing emu in the wild can lay an egg approximately every 72 hours inbreeding season. In contrast, an ostrich in captivity often takes 5 to10 days to lay an egg, and an emu often takes 4 to 8 days to lay an egg.

A slaughter market of ratites will not evolve until sufficient numbersof offspring are produced annually. According to some estimates, inorder for a slaughter market to be maintained, there must be at least250,000 animals available annually. Therefore, a method of enhancingproduction performance will greatly accelerate the growth of the market.Accordingly, there is a need for a composition and method of enhancingproduction performance in birds, and particularly in ratites such asostriches and emus.

In addition to the newly emerging ratite industry, an enormous industryexists which encompasses the more established poultry species, primarilyegg-type chickens (Single Comb White Leghorns), meat-type chickens(broilers), turkeys, ducks, geese, and quail. Worldwide demand forpoultry meat and egg products is great and has been steadily increasingduring the last decade.

The following is an attempt to characterize the size and complexity ofthe poultry meat industry in the USA, exclusive of ratite production.For purposes of this discussion, it should be understood that currentpopulation figures (U.S. Census, 1990 data) suggest that there areapproximately 250 million inhabitants of this country. Furthermore, thepopulation of America by the year 2000 is projected to be approximately275 million people which represents an increase of 25 million people.From 1990 to 1995 (using U.S.D.A. projected figures for 1995), percapita consumption of broiler and turkey flesh averaged 76.7 and 18.1pounds, respectively (See Anonymous, Poultry Processing Sourcebook, MeatProcessing, Vol. 33(9): 22-25, (1994) and Bowman, M., Beef and Pork:Competing for the Food Dollar, Meat Processing, Vol. 33(12): 16-25,(1994)). This translates into an average total per capita poultry meatconsumption of 94.8 pounds during the last 5 years.

Also documented is the steady trend of rising poultry meat consumption.For example, broiler per capita consumption has increased approximately28 pounds (from 55.6 pounds in 1985 to 83.5 pounds in 1995). Likewise,turkey per capita consumption has risen approximately 2.5 pounds (from15.9 pounds in 1989 to 18.4 pounds in 1995). This trend of increasedconsumption of chicken and turkey meat products is expected to continuein the future parallel to the anticipated growth in human population(See values cited above). If both predictions are correct, then by theyear 2000, nearly 16.6 million tons of chicken and turkey meat will beconsumed annually in this country alone. The trend of increased poultryconsumption is directly related to the fact that poultry meats areconsidered to be "heart-healthy" foods (low in animal fat content) andpricewise, they favorably compete with the more expensive red-meats(such as beef, pork, and lamb).

Enhancing production performance of chicken and turkey breeder henswould therefore be of significant economic value to an industrycurrently enjoying high growth due to an ever increasing product demand.To satisfy consumer demand and maintain their competitive edge in meatpricing, broiler and turkey breeders will continue to be in the businessof producing as many hatching eggs as possible. Therefore, any methodcapable of increasing egg production by even small amounts wouldgenerate significant economic benefits.

As an example, the scenario of a single broiler breeder hen (per henhoused basis) laying 15 additional eggs during one production cycle(approximately 1 year in length) is as follows. Such a case would, atcurrent market chick prices ($0.16/chick), result in the generation ofan extra $2.00 income from the sale of additional hatchlings and$10-12.00 as proceeds from the added sales of chicken meat generatedfrom the grow-out of these chicks (after considering appropriatedeductions for feed costs and fixed costs). Because the estimatedbroiler breeder hen population is believed to be in excess of 60 millionhens in the United States, economic gain would be approximately$750+million. Including the estimating monies that could be anticipatedby enhancing production performance in turkey breeders (as well as inhens comprising the specialized poultry businesses of ducks, geese andquail, and including the chickens discussed above) it is conservativelyestimated to be between $1-2 billion for the overall economic gain thatmight be realized from the meat-side of the poultry industry, includingall poultry raised for consumption of flesh, excluding ratites.

It should be noted that the poultry industry first developed in thiscountry as primarily a table egg industry. When fowl were subsequentlyselected for enhanced body weight (meat-type chickens), such geneticselection had deleterious effects on egg production rates. In otherwords, egg production proved to be negatively correlated with thegenetic change found after selection for increased body weight.Therefore, the potential to enhance production performance in broilerand turkey breeder hens (meat-type birds) via endocrine manipulation(such as is contemplated in the present invention) should be greater inmeat-type birds than in Single Comb White Leghorns, birds bred forintensity of egg production since the late 1920s. On the other hand,there are 3 to 4 times as many table egg layers, as discussed below,than broiler breeder birds in America. Therefore, even very smallenhancements in production performance in egg-type birds are greatlymagnified when one considers the size of the bird populations that canbe affected.

The following is a discussion of the size and importance of the tableegg industry in this country. Per capita consumption of eggs hasremained reasonably stable from 1989 to 1992 and ranged between 30.0 to30.4 pounds (See Table 653: Per capita consumption of major foodcommodities, Unites States, 1984-92, Agricultural Statistics, 1993,U.S.D.A. Natl. Agri. Stat. Ser., U. S. Govt. Printing Office,Washington, D. C., p. 457; 1993 and 1994 data unavailable). Stagnanttable egg consumption rates in America are likely directly associatedwith public concern with consumption of egg yolks which are perceived tobe high in cholesterol content.

Despite stable per capita egg consumption rates, the numbers of tableegg layers have steadily increased from 228.8 million hens in 1990 to240.7 million hens in 1994 (See Bell, D. D., University of CaliforniaMonthly Statistical Report, Table 28: Table egg layers: Number on farmsduring month, 1980-93, UC Riverside). This trend of increasing numbersof laying hens may simply reflect a need for more eggs to satisfyincreased population numbers, increased export of eggs and egg products,or yet other unidentified factors. Whatever the reason, because almost80% of the world's eggs are produced outside the Americas, this suggeststhat any positive effects that can be realized within the United Statestable egg market may be multiplied by a factor of five to achieveworldwide economic significance. However, if one restricts calculationssolely to the United States, and one holds per capita consumption steadyat approximately 30.2 pounds, and accepts the projection that ourpopulation will increase to 275 million people in the next 5 years, thenby the year 2000, nearly 4.2 million tons of chicken eggs will beproduced annually in this country alone.

A dollar value for the enhancement of egg production in table egg layersis difficult to derive due to the existence of many complex features ofa Leghorn's production cycle that affect decision making (e.g., cost ofreplacement pullets versus the use of molting, deleterious effects ofmolting on post-molt performance, loss of egg sales during molting,etc.). However, it would not be unreasonable to conclude that anincrease in egg lay in egg-type chickens, on a per hen housed basis ofonly half the magnitude of that discussed above for broiler breeders,would translate into at least a multi-million dollar economic benefit.

The phrase "enhancing production performance" is understood by those ofordinary skill in the art to denote an increase in one or more of thefollowing in female birds: accelerated onset of egg lay; acceleratedonset of maximum egg production; prolonged persistence of egg lay;increased intensity of egg lay; or increased total lifetime egg lay. Thephrase also includes improved feed conversion ratios; improved egg shellquality; or improved resistance to adverse laying conditions such asheat stress, overcrowding, poor nutrition, and noise. The phrase meansan increase in one or more of the following in males: accelerated onsetof puberty or production of sperm; accelerated onset of maximum spermproduction; increased persistence of sperm production; increasedintensity of sperm production (sperm count); increased ejaculate volume;improved sperm viability; increased testosterone production; orincreased libido.

Recently, the hormone inhibin has been studied as a potential means forincreasing ovulatioin in mammals. Inhibin is a peptide hormone primarilyproduced by the gonads, and more particularly by growing follicles andtestes. In mammals, it functions as an inhibitory feedback regulator ofpituitary follicle-stimulating hormone ("FSH") secretion. Whileinhibin's existence was first postulated over 60 years ago, its chemicalisolation was only recently achieved.

Mammalian inhibin is a dimeric protein hormone which is composed of anα-subunit (molecular weight 18,000) and a β-subunit (molecular weight14,000). The α-subunit is unique to inhibin as dimers of the β-subunitform activin, a hormone which releases FSH from the pituitary gland. Theβ-subunit exists in two forms (β_(A) and β_(B)), which are distinct butquite similar. Therefore, depending on the β-subunit involved, inhibinexists as inhibin-A or inhibin-B. Both subunits α and β, when joined bydisulfide bonds, are required for biological activity in suppressingfollicle-stimulating hormone ("FSH") secretion from the pituitary. Theamino acid sequence of the α-subunit of inhibin exhibits approximately80-90% similarity among the porcine, bovine, human, murine, and domesticchicken species. Excellent reviews on the isolation, production, assay,and biological actions of inhibin are available in Risbridger et al.,Current Perspectives of Inhibin Biology, Acta Endocrinologica (Copenh),122:673-682, (1990); and Rivier, C., et al., Studies of the InhibinFamily of Hormones: A Review, Hormone Research, 28: 104-118 (1987),which are hereby incorporated by reference.

In mammals and birds, FSH plays a role in follicular growth anddevelopment, while luteinizing hormone ("LH") is believed to induceovulation. Several brain and gonadal factors (peptide and steroidhormones) interact to control gonadotropin hormone release. Of thesefactors, gonadotropin-releasing hormone ("GnRH") and inhibin exertopposite controls on pituitary FSH secretion in mammals.Gonadotropin-releasing hormone is a brain decapeptide which acts tostimulate FSH and LH secretion, while inhibin is a gonadal protein whichapparently acts to selectively inhibit FSH secretion in mammals.

A basic knowledge of the avian ovulatory process is needed to understandthe role of inhibin in the endocrine control of ovulation in birds.Growing follicles on the functionally mature ovary of the domestic henexist in a distinct size hierarchy. A typical ovary contains four to sixlarge, two to four centimeter in diameter, yolk-filled follicles (F₁ toF₄, F₆), accompanied by a greater number of smaller, two to tenmillimeter, yellow follicles, and numerous very small white follicles.The largest preovulatory follicle (F₁) is destined to ovulate the nextday, the second largest (F₂) on the following day (approximately 26hours later), and so on. The control of follicular recruitment anddevelopment within this hierarchy is poorly understood. Pituitarygonadotropin involvement has been proven, yet the role of inhibin in thecontrol of avian gonadotropin secretion and control of ovulation remainsunclear.

A recent strategy to induce hyper-ovulation in mammalian species hasbeen the development of methods which involve neutralization ofendogenous inhibin activity. For example, the active immunization ofmammals against various inhibin-containing compounds has been studied.Immunoneutralization of inhibin has been associated with increasedovulation rates in heifers, sheep, gilts, and rats.

Accelerated ovulation rates found in mammals vaccinated with antigenicinhibin preparations is thought to be a consequence of elevated plasmaFSH levels which lead to enhanced ovarian follicular development. Avariety of antigens have been used as vaccines in studies whichdemonstrated an elevation in ovulation rate of mammals. Some of theantigens tested in mammals include: recombinant DNA derived fragments ofthe inhibin α-subunit (Wrathall et al., Effects of active immunizationagainst a synthetic peptide sequence of the inhibin α-subunit on plasmagonadotrophin concentrations, ovulation rate and lambing rate in ewes,J. Reprod. Fert., 95:175-182, 1992; and Meyer et al., Antiserum to anInhibin Alpha-Chain Peptide Neutralizes Inhibin Bioactivity andIncreases Ovulation Rate in Sheep, Scientific Journal Series of theMinnesota Agric. Exp. Sta., paper No. 17,103, 1991), synthetic peptidereplicas of the N-terminal sequence of bovine inhibin α-subunit coupledto ovalbumin (Glencross et al., Effect of active immunization of heifersagainst inhibin on plasma FSH concentrations, ovarian folliculardevelopment and ovulation rate, Journal of Endocrinology, 134, 11-18,1992), synthetic peptide sequences of bovine inhibin α-subunitconjugated to human serum albumin (Morris, et al., Effect ofimmunization against synthetic peptide sequences of bovine inhibinα-subunit on ovulation rate and twin-calving rate in heifers, Journal ofReproduction and Fertility, 97:255-261, 1993), and partially purifiedinhibin from bovine follicular fluid (Morris et al., Effect ofImmunizing Prepuberal Lambs of Low and High Ovulation Rate Genotypeswith Inhibin Partially Purified From Bovine Follicular Fluid,Theriogenology, Vol. 35 No. 2, 1991).

Despite conflicting data on how levels of FSH fluctuated during theovulatory cycle, in all cycling mammals studied, immunoneutralization ofendogenous inhibin consistently enhanced ovarian follicular developmentand ovulation rate, regardless of the antigen used or the mammalianspecies challenged.

As stated above, inhibin involvement in the regulation of reproductivefunction in avian species remains unclear. Thus far, published reportshave been restricted to the reproductive function of inhibin in domesticfowl. The bulk of this literature supports the theory that inhibinlikely exerts parallel physiological roles in fowl to those documentedin mammals: in hens, inhibin may serve as a regulator of follicularrecruitment and/or development. However, in birds, inhibin's involvementin the control of ovulation rate may or may not be through suppressionof pituitary FSH secretion. For example, although low egg producing henshave been found to have higher levels of inhibin in plasma and thegranulosa cell layers of preovulatory follicles than high egg producinghens, no difference has been found in plasma FSH levels associated withthe rate of egg laying. Wang et al., Increase in Ovarian α-Inhibin GeneExpression and Plasma Immunoreactive Inhibin Level is Correlated with aDecrease in Ovulation Rate in the Domestic Hen, General and ComparativeEndocrinology, 91, 52-58, (1993). This reference, therefore, suggeststhat in hens the ovulation rate-related changes in inhibin α-subunitgene expression and plasma immunoreactive inhibin levels do not directlyaffect ovulation rate through a modulation of plasma FSH levels.Further, in Johnson, P. A., Inhibin in the Hen, Poultry Science.72:955-958, (1993), a bovine inhibin RIA system was used to successfullyassess immunoreactive inhibin in the plasma of hens, however nosignificant peak of immunoreactive inhibin was detected throughout theovulatory cycle in spite of a preovulatory surge of LH. Accordingly, therole of inhibin in folliculogenesis in birds remains unclear.

Recently, the α-subunit of chicken inhibin was successfully cloned andsequenced. Wang and Johnson, Complementary Deoxyribonucleic Acid Cloningand Sequence Analysis of the α-subunit of Inhibin from Chicken OvarianGranulosa Cells, Biology of Reproduction, 49, 1-6, (1993), which isincorporated herein by reference in its entirety. Comparison of theavian inhibin sequence to known mammalian inhibin α-subunit sequencesshowed an 86-89% homology. Northern blot analysis using two isolatedprobes (cINA₆ and cINA₁₂) revealed that the inhibin α-subunit isexpressed in chicken ovarian granulosa cells but not in chicken brain,kidney, liver or spleen tissues.

Therefore, the biology of inhibin in birds remains poorly understood,and the responses of birds to challenges with antigenic inhibin has notyet been attempted or monitored. Accordingly, as the ratite market issubstantially limited by the sub-optimal production performance of manyostriches, emus, and rhea, what is needed is a composition and methodfor enhancing the production performance of these birds.

Further, the improvement of the production performance of all poultry isneeded to increase the amount of poultry produced for consumption and toimprove the efficiency of such production, or feed conversion ratio.Accordingly, there remains a need for a composition and method ofimproving or enhancing production performance for all poultry, includingchickens, turkeys, ducks, quail, and geese, among others.

There also is a need for a composition and method of enhancingproduction performance in exotic birds, such as the Psittaciformes.Psittaciformes include parrots, and are a monofamilial order of birdsthat exhibit zygodactylism and have a strong hooked bill. A parrot isdefined as any member of the avian family Psittacidae (the single familyof the Psittaciformes), distinguished by the short, stout, stronglyhooked beak.

The need for a composition and method for enhancing productionperformance is not limited to birds. There remains a need for aneffective composition and method for enhancing production performance inmany animals. For example, there is a continued need for enhancingproduction performance in most animals that are raised agriculturally,such as pigs, cows, and sheep. There is also a continued need ofenhancing production performance in fur bearing animals such as mink,fox, otter, ferret, raccoons, and in rodents such as rats, mice,gerbils, and hamsters used as pets and as laboratory research subjects,and there is an increased need for other animals whose hides are usedfor decorative purposes.

Also, a composition and method for enhancing production performance isalso needed to increase the population of many animals such as exotic orendangered species to avoid their extinction. There is further acontinued need for enhancing production performance in animals used forracing, entertainment, or showing (competitions) such as horses, dogs,cats, zoo animals, and circus animals. As shown by the increased demandsfor infertility treatment of humans, there is also a need for enhancingproduction performance in humans. Accordingly, there remains a need fora composition and method for enhancing production performance in manyanimals.

SUMMARY OF THE INVENTION

The present invention relates, in general, to a method of enhancing theproduction performance of animals, by administering to the animal aheterologous protein comprised of inhibin protein, or a fragmentthereof, and a carrier protein. The present invention also relates to amethod of enhancing the production performance of animals, byadministering to the animal a fusion gene product comprising a geneencoding for the expression of alpha-subunit inhibin protein, or afragment thereof, and a gene encoding for the expression of a carrierprotein. An effective amount of the heterologous protein or fusion geneproduct is administered to an animal such that an immunological responseoccurs in the animal against the heterologous protein. It is to beunderstood that the method of the present invention enhances productionperformance of animals that produce inhibin. Preferably, the animal is abird. More preferably, the bird is a chicken. Another preferred bird isa ratite, such as, an emu, an ostrich, a rhea, or a cassowary.

The present invention further relates to the above heterologous proteinand fusion gene product, and to methods of producing the same. Moreparticularly, the present invention is directed to a composition andmethod for making a heterologous protein comprising inhibin, or afragment thereof, and a carrier protein. The inhibin protein, orfragment thereof, can be avian inhibin, mammalian inhibin, piscineinhibin, or reptilian inhibin. The carrier protein, includes, but is notlimited to, maltose binding protein, thyroglobulin, keyhole limpethemocyanin, or bovine serum albumin, among others. The preferred carrierprotein is maltose binding protein.

The heterologous protein can be either inhibin conjugated to the carrierprotein or inhibin fused to the carrier protein. The method of producingthe fused heterologous protein comprises inserting cDNA which is encodedfor expressing inhibin, or a fragment thereof, into a vector whichcontains coding information for the production of a carrier protein.After inserting the vector into an expression system, the fusedheterologous protein is expressed by the system. Preferably, theheterologous protein is comprised of ratite inhibin, such as ostrichinhibin, emu inhibin, and rhea inhibin. Another preferred heterologousprotein is comprised of chicken inhibin.

The present invention is also directed to a method of enhancingproduction performance in animals via the administration of theheterologous protein of the present invention which comprises inhibinprotein, or a fragment thereof, and a carrier protein. In oneembodiment, the method comprises administering an effective amount ofthe protein to a female animal. In another embodiment, the methodcomprises administering an effective amount of the protein to a maleanimal. Preferably, an immunological response occurs in the animaldirected against the heterologous protein. More preferably, theimmunological response which occurs in the animal is also directedagainst the inhibin protein produced by the animal (endogenous inhibin).

The present invention is also directed to a fusion gene productcomprising a gene encoded for the expression of alpha-subunit inhibinprotein, or a fragment thereof, and a gene encoded for the expression ofa carrier protein. The gene encoded for the expression of inhibinprotein, or fragment thereof, may be encoded to express avian inhibin,mammalian inhibin, piscine inhibin, or reptilian inhibin. The geneencoded for the expression of a carrier protein may be encoded toexpress maltose binding protein or bovine serum albumin, among others.The preferred gene encoded to express a carrier protein is encoded toexpress maltose binding protein.

The present invention also relates to a method of enhancing theproduction performance of animals, by administering to the animal afusion gene product comprising a gene encoded for the expression ofalpha-subunit inhibin protein, or a fragment thereof, and a gene encodedfor the expression of a carrier protein. More particularly, the presentinvention further encompasses gene therapy methods whereby DNA sequencesencoding inhibin, or fragments thereof, and a carrier protein areintroduced into an animal. The fusion gene product of the presentinvention may be administered directly to the animal, or it may beadministered in a vector, or in a cell containing a vector having thefusion gene product therein.

The method of the present invention enhances production performance infemale animals which produce inhibin, such as mammals, reptiles, fish,and birds. More particularly, this method enhances productionperformance in female galliformes and ratites. More particularly, thismethod enhances production performance in chickens, turkeys, ostriches,emus, and rhea. This method also enhances production performance inturtles, including endangered turtle species. Unexpectedly, the methodof the present invention increases the onset of puberty or egg lay inanimals. Also, the method of the present invention unexpectedlyaccelerates the onset of maximum egg lay in an animal. Further, themethod of the present invention increases the intensity of egg lay of ananimal. Further still, the method of the present invention surprisinglyprolongs the persistence of maximum egg lay in animals. Still further,the method unexpectedly increases the lifetime total egg lay of ananimal. In avians, the method of the present invention also improves thefeed conversion ratio of a bird. Also, the method of the presentinvention unexpectedly reduces or eliminates the effect of adverselaying conditions on egg lay rates of animals exposed to suchconditions. Such adverse conditions include elevated temperatures,overcrowding, poor nutrition, and noise.

Surprisingly, the method of the present invention also improvesproduction performance in male animals which produce inhibin, such asmammals, reptiles, and birds. More particularly, the method of thepresent invention increases testosterone levels in male animals.Similarly, the method of the present invention increases the onset ofpuberty or sperm production in male animals. Also, the method of thepresent invention accelerates the onset of maximum sperm production in amale animal. Further, the method of the present invention unexpectedlyincreases the intensity of sperm production (sperm count) by a maleanimal. Further still, the method of the present invention prolongs thepersistence of maximum sperm production in animals. Also, the methodimproves sperm viability in animals. Still further, the methodunexpectedly reduces or eliminates the effect of adverse conditions onsperm production of animals exposed to such conditions. Such adverseconditions include elevated temperatures, overcrowding, poor nutrition,and noise. The method of the present invention also surprisinglyincreases the libido, and therefore, the reproductive potential, of amale bird.

As stated above, the method of the present invention is used to enhanceproduction performance of any animal that produces inhibin, including,but not limited to, most animals that are raised agriculturally, such aspigs, cows, sheep, turkeys, quail, ducks, geese, chickens, and fish; infur bearing animals such as mink, fox, otter, ferret, rabbits andraccoon; laboratory animals such as rats, mice, gerbils, and guineapigs; for animals whose hides are used for decorative purposes such asalligators and snakes; exotic or endangered species; animals used forracing, entertainment, or showing (competitions) such as horses, dogs,cats, zoo animals, and circus animals; and humans. Additional aviansthat the method of the present invention enhances production performancethereof include ratites, psittaciformes, falconiformes, piciformes,strigiformes, passeriformes, coraciformes, ralliformes, cuculiformes,columbiformes, galliformes, anseriformes, and herodiones. Moreparticularly, the method of the present invention may be used to enhanceproduction performance of an ostrich, emu, rhea, chicken, turkey, ducks,geese, quail, partridge kiwi, cassowary, parrot, parakeet, makaw,falcon, eagle, hawk, pigeon, cockatoo, song bird, jay bird, blackbird,finch, warbler, canary, toucan, mynah, or sparrow.

Accordingly, it is an object of the present invention to provide aninhibin composition which induces an immunological response in an animalupon its administration to an animal.

It is yet another object of the present invention to provide aheterologous protein comprising inhibin protein, or a fragment thereof,and carrier protein.

It is a further object of the present invention to provide a compositioncomprising a fused heterologous protein comprised of inhibin, or afragment thereof.

It is further an object of the present invention to provide a method forproducing a fused heterologous protein comprising inhibin protein, or afragment thereof, and a carrier protein.

It is yet another object to provide a fusion gene product comprising agene encoded for the expression of alpha-subunit inhibin protein, or afragment thereof, and a gene encoded for the expression of a carrierprotein,

It is a further object of the present invention to produce animmunological response directed against the heterologous protein of thepresent invention by direct injection of the fused gene product of thepresent invention into an animal.

Yet another object of the invention is to provide compositions andmethods useful for gene therapy for the modulation of inhibin levels.

It is another object of the present invention to provide a method forenhancing production performance in animals.

It is an object of the present invention to provide a method forenhancing production performance in birds.

It is a further object of the present invention to provide a method forenhancing production performance in ratites.

It is also an object to provide a method for enhancing productionperformance in chickens.

It is yet another object to provide a method for enhancing productionperformance in reptiles.

Is another object of the present invention to provide a method forenhancing production performance in mammals.

Is another object of the present invention to provide a method forenhancing production performance in fish.

It is further an object of the present invention to provide a method forenhancing production performance in humans.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an SDS-PAGE gel wherein A is ostrich anti-(chickeninhibin-maltose binding protein) antibodies, B is plasmid pMAL™-c vectorstandard, C is protein molecular weight standards, D is the actualpMAL™-c vector used in the preparation of the fused heterologousprotein, E is the purified fused chicken inhibin-maltose binding protein(heterologous protein) of the present invention, and F is eluent from apurification that was not loaded with the heterologous protein.

FIG. 2 is an illustration of the effect of inhibin α-subunitimmunoneutralization on hen-day egg production ("HDEP") in JapaneseQuail using maltose binding protein fused with the protein encoded bycINA₅₂₁. More particularly, FIG. 2 is a graphic representation of thedata in Example 8.

DETAILED DESCRIPTION

The present invention relates, in general, to a method of enhancing theproduction performance of animals, by administering to the animal aheterologous protein comprised of inhibin protein, or a fragmentthereof, and a carrier protein. The present invention also relates to amethod of enhancing the production performance of animals, byadministering to the animal a fusion gene product comprising a geneencoded for the expression of alpha-subunit inhibin protein, or afragment thereof, and a gene encoded for the expression of a carrierprotein.

An effective amount of the heterologous protein or fusion gene productis administered to an animal such that an immunological response occursin the animal against the heterologous protein. It is to be understoodthat the method of the present invention enhances production performanceof animals that produce inhibin. Preferably, the animal is a bird. Morepreferably, the bird is a chicken. Another preferred bird is a turkey.Yet another preferred bird is a ratite, such as, an emu, an ostrich, arhea, or a cassowary. The present invention further relates to the aboveheterologous protein and fusion gene product, and to a method ofproducing the same.

After the following definitions, the composition of the presentinvention is described in detail, followed by a detailed description ofthe methods of the present invention.

Definitions

The term "bird" or "fowl," as used herein, is defined as a member of theAves class of animals which are characterized as warm-blooded,egg-laying vertebrates primarily adapted for flying. The term "Ratite,"as used herein, is defined as a group of flightless, mostly large,running birds comprising several orders and including the emus,ostriches, kiwis, and cassowaries. The term "Psittaciformes", as usedhere, include parrots, and are a monofamilial order of birds thatexhibit zygodactylism and have a strong hooked bill. A "parrot" isdefined as any member of the avian family Psittacidae (the single familyof the Psittaciformes), distinguished by the short, stout, stronglyhooked beak. The term "chicken" as used herein denotes both chickensused for egg production, such as Single Comb White Leghorns, andchickens raised for consumption, or broilers.

The term "egg" is defined herein as a large female sex cell enclosed ina porous, calcarous or leathery shell, produced by birds and reptiles."Egg production by a bird or reptile", as used herein, is the act of abird laying an egg, or "oviposition". The term "ovum" is defined as afemale gamete, and is also known as an egg. Therefore, egg production inall animals other than birds and reptiles, as used herein, is defined asthe production and discharge of an ovum from an ovary, or "ovulation".Accordingly, it is to be understood that the term "egg" as used hereinis defined as a large female sex cell enclosed in a porous, calcarous orleathery shell, when it is produced by a bird or reptile, or it is anovum when it is produced by all other animals.

The terms "onset of egg lay", "first egg lay" and "puberty", inreference to birds are used interchangeably herein, and denote when abird lays its first egg. Accordingly, "accelerating the onset" of egglay or puberty in avians, as used herein, denotes inducing an earlierdate of first egg lay than a bird would normally have. Similarly,"puberty" and "onset of sperm production" in males are usedinterchangeably.

The phrases "enhancing production performance", "improving productionperformance" and "increasing production performance" are usedinterchangeably to denote an improvement in one or more of the followingareas: accelerated onset of puberty (egg lay or ovulation in females;sperm production in males); accelerated onset of maximum egg lay orovulation in females or accelerated onset of maximum sperm production inmales; increased intensity of production of eggs in females, or of spermin males; prolonged persistence of egg lay in females or of spermproduction in males; increased total lifetime egg lay or ovulation infemales; improved feed conversion ratios; improved egg shell quality;improved resistance to adverse conditions such as elevated temperatures,overcrowding, poor nutrition, and noise; improved sperm viability inmales; increased testosterone production in males; increased ejaculatevolume; and increased libido in males.

The phrase "intensity of egg lay" is known to those of ordinary skill inthe art to denote frequency of egg lay.

The phrase "lifetime total egg lay" of a bird is defined as the totalnumber of eggs laid by a bird during its entire life span. The phrase"hen day egg production" or "HDEP", as used herein, is defined as thenumber of eggs laid by a particular group of hens per day.

The phrase "accelerated onset of maximum egg lay" or "accelerated onsetof maximum egg production" as used herein, denotes that the period oftime from birth to when the animal lays eggs or ovulates at 50% of itspeak lay rate or ovulation rate, is shorter than the normal period oftime from birth to maximum egg lay.

It is to be understood that a method of "decreasing cholesterol levels"of an egg, as used herein, denotes a method of inducing a bird to layone or more eggs having a lower cholesterol content than the averagecholesterol content of eggs laid by birds of the same species.

In contrast to the term bird or fowl, the term "mammal", as used herein,is defined as a member of the class Mammalia which is a large class ofwarm-blooded vertebrates containing animals characterized by mammaryglands, a body covering of hair, three oscicles in the middle ear, amuscular diaphragm separating the thoracic and abdominal cavities, redblood cells without nuclei, and embryonic development in the allantoisand anmion.

The term "reptile", as used herein, is defined as any member of theclass Reptilia which is a class of terrestrial vertebrates whichcharacteristically lack hair, feathers, and mammary glands, their skinbeing covered with scales, they have a three chambered heart, and theirpleural and peritoneal cavities are continuous.

A heterologous protein, as used herein, is defined as a proteincomprised of inhibin protein, or a fragment thereof, and a carrierprotein. It is to be understood that the terms "inhibin" and "fragmentof inhibin" are used interchangeably in the heterologous proteincomposition, the method of making the heterologous protein, and themethod of using the heterologous protein of the present invention.

It is also to be understood that "cINA₅₂₁ ", as used herein, denotes a521 base pair sequence (SEQ ID NO:l). cINA₅₂₁ codes for a portion of thealpha-inhibin subunit of a chicken, represented by SEQ ID NO:2. As usedherein, "MBP-cINA₅₂₁ " is the heterologous protein that is expressedfrom a recombinant host cell, after cloning cINA₅₂₁ into a recombinanthost cell and expressing a fused heterologous protein comprising maltosebinding protein ("MBP") and the inhibin protein alpha-subunit fragmentencoded by cINA₅₂₁. Preferably, MBP-cINA₅₂₁ is produced in host E. colicells after expression of cloned cINA₅₂₁ using the commerciallyavailable vector pMAL™-c. Accordingly, "cINA21" denotes a nucleotidesequence, and "MBP-cINA₅₂₁ " denotes a fused heterologous protein.

A fused heterologous protein, as used herein, is defined as twodifferent proteins fused together. For example, a protein comprised ofinhibin protein, or a fragment thereof, fused to a carrier protein. Thefused heterologous protein is expressed from an expression systemcomprising a fused gene product which contains a gene encoded for theexpression of inhibin protein, or a fragment thereof, fused to a geneencoded for expression of a carrier protein. "Fused gene product", asused herein, is defined as the product resulting from the fusion of thegene encoded for the expression of inhibin protein, or a fragmentthereof, and the gene encoded for the expression of a carrier protein.

A conjugated heterologous protein, as used herein, is defined as aprotein comprised of inhibin protein, or a fragment thereof, conjugatedto a carrier protein. The conjugated heterologous protein is produced bya chemical reaction which links the inhibin protein to the carrierprotein with a covalent bond.

An immunological response of an animal to a substance which has beenadministered to the animal, as used herein, is defined as thecell-mediated and/or humoral response of an animal which is specificallydirected against the substance.

The term "selectively interact", as used herein, is defined as where twoobjects associate with each other by a covalent bond, a noncovalentbond, a hydrogen bond, electrostatically, a receptor-ligand interaction,a enzyme-substrate interaction, or by other binding or attachment means.The association is selective in that the two objects interact in aspecific manner, in a specific position, or only with each other.

Inhibin Compositions

The present invention relates in general to a composition used in amethod of enhancing production performance in animals, including birds.The composition is comprised of a heterologous protein comprisinginhibin protein, or a fragment thereof, and a carrier protein. Theinhibin can be inhibin from any species of animal that produces inhibin.The inhibin includes, but is not limited to, bird inhibin, mammalinhibin, reptile inhibin, amphibian inhibin, or fish inhibin, amongothers. More specifically, the mammal inhibin includes, but is notlimited to, cow inhibin, human inhibin, horse inhibin, cat inhibin, doginhibin, rabbit inhibin, sheep inhibin, mink inhibin, fox inhibin, otterinhibin, ferret inhibin, raccoon inhibin, donkey inhibin, rat inhibin,mouse inhibin, hamster inhibin, and pig inhibin. The bird inhibinincludes, but is not limited to, ostrich inhibin, emu inhibin, rheainhibin, cassowary inhibin, kiwi inhibin, turkey inhibin, quail inhibin,chicken inhibin, duck inhibin, goose inhibin, and inhibin from membersof the order psittaciformes.

A preferred inhibin is avian, or bird, inhibin. A more preferred inhibinis ratite inhibin, such as ostrich, emu, or rhea inhibin. An especiallypreferred inhibin is ostrich inhibin. Another preferred inhibin ischicken inhibin. Most preferably, the heterologous protein of thepresent invention comprises alpha-subunit inhibin protein, or a fragmentthereof, and a carrier protein.

The inhibin, or fragment thereof, can be isolated from animal fluids,expressed from genetically engineered cells in an expression system, orsynthetically produced from a series of chemical reactions. Moreparticularly, the fragment of inhibin includes, but is not limited tothe following compositions: α-subunit inhibin; β-subunit inhibin;recombinant DNA derived fragments of α-subunit inhibin or β-subunitinhibin; synthetic peptide replicas of fragments of α-subunit inhibin orβ-subunit inhibin; synthetic peptide replicas of the N-terminal sequenceof α-subunit inhibin or β-subunit inhibin; fragments of partiallypurified inhibin from follicular fluid; fragments of endogenousα-subunit inhibin or β-subunit inhibin; and fragments of exogenousα-subunit inhibin or β-subunit inhibin. As stated above, it is mostpreferable that the fragment of inhibin is alpha (α)-subunit inhibin, ora fragment thereof. By inhibin, it is understood by one of ordinaryskill in the art to encompass inhibin with amino acid substitutions thatmight render it more immunogenic, or more active at a receptor.

The inhibin in the heterologous protein is either fused to or conjugatedwith the carrier protein as described below. Where the inhibin is fusedto the carrier protein, the heterologous protein is a "fusedheterologous protein". Where the inhibin is conjugated to the carrierprotein, the heterologous protein is a "conjugated heterologousprotein". A preferred heterologous protein is a fused heterologousprotein.

The identity of the carrier protein in the heterologous protein is not acritical aspect of the present invention. Any carrier protein known inthe art can be used in the heterologous protein. The carrier proteinsthat can be used in the present invention include, but are not limitedto the following group: maltose binding protein "MBP"; bovine serumalbumin "BSA"; keyhole limpet hemocyanin "KLH"; ovalbumin; flagellin;thyroglobulin; serum albumin of any species; gamma globulin of anyspecies; syngeneic cells; syngeneic cells bearing Ia antigens; andpolymers of D- and/or L- amino acids. A preferred carrier protein isMBP. Another preferred carrier protein is BSA if the heterologousprotein will not be administered to a cow or horse. Yet anotherpreferred carrier protein is ovalbumin if the heterologous protein willnot be administered to a bird. The most preferred carrier protein isMBP. It is preferred that the carrier protein is immunogenic to theanimal that it will be administered to.

The present invention also relates to a method of producing theconjugated heterologous protein of the present invention. Methods ofproducing conjugated proteins are well known in the art. Methods ofconjugating proteins to proteins are fully described in Antibodies, ALaboratory Manual, edited by Ed Harlow & David Lane, Coldspring HarborLab (1988), which is incorporated herein by reference. Additionalmethods of producing conjugated heterologous proteins, includingconjugation reagents, such as dialdehydes, carbodiimides, bisdiazotizedbenzidine and others, carrier proteins, and immunization schedules aredescribed in detail in Chapter 38, pp. 605-618 and Chapter 42, pp.665-678, in Section VI, "Preparation of Antibodies" in NeuroendocrinePeptide Methodology, edited by P. Michael Conn, Academic Press, NewYork, 1989, which is incorporated herein by reference.

Although conjugated proteins may be used in the methods of the presentinvention, fusion proteins are preferred. More particularly,heterologous proteins that are fused yield a homogeneous product,wherein the different segments of the proteins are always fused in thesame position, and the same amount of the segments of the proteins arefused. Also, fused heterologous proteins can be produced uniformly,inexpensively, and in large quantities. In contrast, conjugatedheterologous proteins are not as uniform as fused proteins. For example,depending on what proteins are being conjugated, the conjugationreaction may yield a mixture of proteins having one or moreconjugations, proteins having conjugations in different locations, orproteins that remain unconjugated. Further, some of the conjugations mayrender the heterologous protein sterically hindered for its intended use(e.g., the immunogenic portion of the protein is sterically hindered).Also, conjugation reaction conditions and reagents may degrade theproteins produced therein. For example, glutaraldehyde is commonly usedin conjugation reactions, and it modifies the conformation of proteins.Further, conjugated proteins are more expensive to produce in largequantities than fused proteins.

The present invention is also directed to a fusion gene productcomprising a gene encoded for the expression of alpha-subunit inhibinprotein, or a fragment thereof, and a gene encoded for the expression ofa carrier protein. The gene encoded for the expression of inhibinprotein, or fragment thereof, may be encoded to express avian inhibin,mammalian inhibin, fish inhibin, or reptilian inhibin. The gene encodedfor the expression of a carrier protein may be encoded to expressmaltose binding protein or bovine serum albumin, among others. Thepreferred gene encoded to express a carrier protein is encoded toexpress maltose binding protein. The fusion gene product and the methodof making the fusion gene product are described more fully below.

Briefly described, the method of producing the fused heterologousprotein of the present invention is comprised of the steps of insertinga fusion gene product into a coding region of a plasmid, transformationinto a host cell with the plasmid, and expressing the fused heterologousprotein from the host cell by methods well known in the art. Moreparticularly, the method of producing the fused heterologous proteincomprises inserting cDNA which is encoded for expressing inhibin, or afragment thereof, into a vector which contains coding information forthe production of a carrier protein. After inserting the vector into anexpression system, the fused heterologous protein is expressed by thesystem.

Many methods of making fused heterologous proteins are known in the art.Therefore, any method known in the art can be used to produce the fusedheterologous protein of the present invention. Many commerciallyavailable vector kits and expression systems can be used to prepare thefused heterologous protein of the present invention. An example of sucha commercially available vector kit and expression system is pMAL™-c ofNew England Biolabs, Beverly, Mass. Cytoplasmic expression of the fusedheterologous protein occurs in the pMAL™-c system. The method ofproducing the fused heterologous protein of the present invention from apMAL™-c kit is fully described below in Examples 1 and 2. Other sourcesof vector kits and expression systems which can be used to produce thefused heterologous protein of the present invention include, but are notlimited to: Pharmacia Biotech of Piscataway, N.J.; and Clonetech, ofPalo Alto, Calif.

The present invention further relates to a fusion gene productcomprising a gene encoded for the expression of inhibin protein, or afragment thereof, and a gene encoded for the expression of a carrierprotein. The inhibin gene can be from any species of animal thatproduces inhibin. The inhibin gene can be a bird inhibin gene, a mammalinhibin gene, a reptile inhibin gene, an amphibian gene, or a fish gene,among others. More specifically, the mammal inhibin gene includes, butis not limited to, bovine inhibin gene, human inhibin gene, equineinhibin gene, cat inhibin gene, dog inhibin gene, sheep inhibin gene,mink inhibin gene, fox inhibin gene, otter inhibin gene, ferret inhibingene, raccoon inhibin gene, rat inhibin gene, mouse inhibin gene,hamster inhibin gene, donkey inhibin gene, and pig inhibin gene. Thebird inhibin gene includes, but is not limited to, an ostrich inhibingene, an emu inhibin gene, a rhea gene, a cassowary inhibin gene, a kiwiinhibin gene, a turkey inhibin gene, a quail inhibin gene, a chickeninhibin gene, an inhibin gene from any member of the orderpsittaciformes, an inhibin gene from any falconiformes, an inhibin genefrom any piciformes, an inhibin gene from any strigiformes, an inhibingene from any coraciformes, an inhibin gene from any ralliformes, aninhibin gene from any passeriformes, an inhibin gene from anycuculiformes, an inhibin gene from any columbiformes, an inhibin genefrom any galliformes (domestic fowl), an inhibin gene from anyanseriformes (geese, ducks, other water fowl), an inhibin gene from anyherodiones, and an inhibin gene from any of the following birds: falcon,eagle, hawk, pigeon, parakeet, cockatoo, makaw, parrot, canary, mynah,toucan, and perching bird (such as, song bird, jay, blackbird, finch,warbler, and sparrow).

A preferred inhibin gene is a bird inhibin gene. A more preferredinhibin gene is a ratite inhibin gene. An especially preferred inhibingene is an ostrich inhibin gene. Another preferred inhibin gene is anemu inhibin gene. Yet another preferred inhibin gene is a rhea inhibingene. Another preferred inhibin gene is a chicken inhibin gene.

The chicken inhibin α-subunit cDNA clone (cINA6; Wang and Johnson,Complementary Deoxyribonucleic Acid Cloning and Sequence Analysis of theα-subunit of Inhibin from Chicken Ovarian Granulosa Cells, Biology OfReproduction, 49: 453-458, 1993), which is hereby incorporated byreference in its entirety, inserted into the EcoR 1 site of Bluescript(Stratagene, La Jolla, Calif.), was obtained as a gift of P. A. Johnson(Cornell University). The cINA6 clone specifically hybridized to ostrichgenomic DNA in Southern assays indicating significant DNA homologybetween these two species (Chouljenko et al., Expression andpurification of chicken α-inhibin as a fusion protein with the E. colimaltose binding protein, Poultry Science, 73(Suppl. 1): 84, 1994). A DNAfragment ("cINA₅₂₁ ") was excised from the cINA6 clone using Pst Idigestion. The cINA₅₂₁ DNA fragment encompassed most of the maturechicken inhibin α-subunit. Although cINA₅₂₁ was excised from the cINA6clone reported in Wang and Johnson, the sequence obtained, namely SEQ IDNO: 1, differs from the DNA sequence published in Wang and Johnson.

The ostrich inhibin α-subunit sequence was obtained by polymerase chainreaction (PCR) methods that are well known in the art. Moreparticularly, the primers were constructed based on the sequencereported in Wang, and were used in a PCR reaction with ostrich genomicDNA: 5'-CTCAGCCTGCTGCAGCGCCC-3'(SEQ ID NO: 3); and5'-GTGTCGACCGCGCGACGCCGAC-3'. More particularly, the above primerscorrespond to base pairs 778 to 797 and 1347 to 1326, respectively, ofthe chicken inhibin α-subunit cDNA clone, cINA₆, reported in Wang andJohnson. The PCR-product was digested with Pst1 endonuclease andsubcloned into commercially available vector PUC19 (New EnglandBiolabs). The sequence of the ostrich Pst1 fragment inhibin gene isidentical to the corresponding portion of the chicken alpha inhibin.

As stated above, it is to be understood that the carrier protein is nota critical aspect of the present invention. Therefore, a gene encoded toexpress any carrier protein can be used in the present invention. Thecarrier protein gene includes, but is not limited to, genes encoded forexpressing the following proteins: maltose binding protein "MBP"; bovineserum albumin "BSA"; keyhole limpet hemocyanin "KLH"; ovalbumin;flagellin; thyroglobulin; serum albumin of any species; gamma globulinof any species; and polymers of D- and/or L- amino acids. A preferredcarrier protein gene is a gene encoded to express MBP. Another preferredcarrier protein gene is a BSA gene if the resultant heterologous proteinwill not be administered to a cow or horse. Yet another preferredcarrier protein gene is an ovalbumin gene if the resultant heterologousprotein will not be administered to a bird. The most preferred carrierprotein gene is a gene encoded to express MBP or derivatives thereof.The preferred carrier protein genes code for proteins that will increaseboth the intensity and duration of the host's immune response to theinhibin protein.

The present invention further relates to a method for making a fusiongene product comprising the step of fusing a gene encoded for theexpression of inhibin protein, or a fragment thereof, to a gene encodedfor the expression of a carrier protein. Briefly described, the methodof making the fusion gene of the present invention comprises the stepsof isolating the desired inhibin complementary DNA (cDNA), producingdouble strand inhibin DNA, obtaining double strand carrier protein DNA,and fusing the double strand inhibin DNA to the double strand carrierprotein DNA in a manner such that the fused DNA enables the expressionof a fused heterologous protein comprising the inhibin protein, or afragment thereof, and the carrier protein.

Many methods of isolating genes and making fusion gene products areknown in the art. See, for example, Sambrook, Fritsch & Maniatis,Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, 1989, Vols. I, II, III. Therefore, any method known inthe art can be used to produce the fusion gene product of the presentinvention. Many commercially available vector kits can be used toprepare the fusion gene product of the present invention. An example ofsuch a commercially available vector kit is pMAL™-c of New EnglandBiolabs, Beverly, Mass. The method of producing the fusion gene productof the present invention from a pMAL™-c kit is fully described below inExample 1. Other sources of vector kits which can be used to produce thefused gene product of the present invention include, but are not limitedto: Pharmacia Biotech of Piscataway, N.J.; and Clonetech, of Palo Alto,Calif.

As stated above, the chicken inhibin α-subunit cDNA clone (cINA6)inserted into the EcoR 1 site of Bluescript was obtained as a gift of P.A. Johnson (Cornell University). A DNA fragment ("cINA₅₂₁ ") was excisedfrom the cINA6 clone using Pst I digestion. This fragment (cINA₅₂₁) wascloned in plasmid p-MAL™-c in frame with the maltose binding protein("MBP") and a fusion protein of appropriate size (Lane E; FIG. 1) wasdetected after IPTG (isopropyl β-D-thiogalactopyranoside) induction andSDS-PAGE. The resulting protein conjugate ("MBP-cINA₅₂₁ ") was used asan antigen to immunize pre-pubescent, female Japanese quail (Coturnixcoturnix japonica) against circulating inhibin levels as is fullydescribed in Example 8.

Methods of Enhancing Production Performance

It has been unexpectedly discovered that the composition of the presentinvention enhances the production performance of animals, and inparticular the production performance of birds. Accordingly, the presentinvention is also directed to a method of enhancing productionperformance in animals via the administration of the heterologousprotein of the present invention. In one embodiment, the methodcomprises administering an effective amount of the protein to a femaleanimal such that production performance of the animal is increased. Inanother embodiment, the method comprises administering an effectiveamount of the protein to a male animal such that production performanceof the animal is increased. Preferably, an immunological response occursin the animal directed against the protein. More preferably, theimmunological response which occurs in the animal is also directedagainst the inhibin protein produced by the animal (endogenous inhibin).

More particularly, the method of the present invention comprises theadministration of an effective amount of the heterologous protein of thepresent invention (comprising inhibin, or a fragment thereof, and acarrier protein) to an animal such that the production performance ofthe animal is enhanced. Preferably, the animal is a bird. It is to beunderstood that a "treated" bird is a bird to which the heterologousprotein of the present invention has been administered.

The method of the present invention can be used to enhance productionperformance in any species of female bird that produces inhibin. Thefemale bird includes, but is not limited to, a ratite, a psittaciformes,a falconiformes a piciformes, a strigiformes, a passeriformes, acoraciformes, a ralliformes, a cuculiformes, a columbiformes, agalliformes (domestic fowl), an anseriformes (geese, ducks, other waterfowl), and a herodiones. More particularly, the female bird includes,but is not limited to, an ostrich, emu, rhea, kiwi, cassowary, turkey,quail, chicken, falcon, eagle, hawk, pigeon, parakeet, cockatoo, makaw,parrot, perching bird (such as, song bird, jay, blackbird, finch,warbler, sparrow), and any member of the order psittaciformes. Apreferred bird is a ratite. A more preferred bird is an ostrich. Anotherpreferred ratite is an emu. Yet another preferred ratite is a rhea.Another preferred bird is any member of the order psittaciformes. Yetanother preferred bird is a chicken. Still another preferred bird is aquail. The method of the present invention can also be used toaccelerate the onset of egg lay in species of birds that are endangered.Such endangered birds include, but are not limited to, eagles, hawks,condors, and owls.

The inhibin and the carrier protein in the heterologous proteincomposition of the present invention vary according to what species ofbird the composition will be administered to. It is preferred that avianinhibin and maltose binding protein is used when the composition is tobe administered to a bird. A preferred inhibin is domestic chicken orratite inhibin when the composition is to be administered to a ratite.More preferably, the preferred inhibin is domestic chicken or ostrichinhibin when the composition is to be administered to an ostrich orratite. Another preferred inhibin is domestic chicken or ostrich inhibinwhen the composition is to be administered to a chicken. It is to beunderstood that the inhibin in the heterologous protein need not be fromthe same species to which the heterologous protein will be administered.For example, a heterologous protein that is administered to an ostrichcan be comprised of chicken inhibin and a carrier protein.

It is also to be understood that the composition can further compriseadjuvants, preservatives, diluents, emulsifiers, stabilizers, and otherknown components that are known and used in vaccines of the prior art.Any adjuvant system known in the art can be used in the composition ofthe present invention. Such adjuvants include, but are not limited to,Freund's incomplete adjuvant, Freund's complete adjuvant, polydispersedβ-(1,4) linked acetylated mannan ("Acemannan"), Titermax®(polyoxyethylene-polyoxypropylene copolymer adjuvants from CytRxCorporation), modified lipid adjuvants from Chiron Corporation, saponinderivative adjuvants from Cambridge Biotech, killed Bordetellapertussis, the lipopolysaccharide (LPS) of gram-negative bacteria, largepolymeric anions such as dextran sulfate, and inorganic gels such asalum, aluminum hydroxide, or aluminum phosphate. A preferred adjuvantsystem is Freund's incomplete adjuvant. Another preferred adjuvantsystem is Freund's complete adjuvant.

The heterologous protein composition of the present invention can beadministered to a bird by any means known in the art. For example, thecomposition can be administered subcutaneously, intraperitonealy,intradermally, or intramuscularly. Preferably, the composition isinjected subcutaneously. The composition can be administered to the birdin one or more doses. Preferably, the composition is administered to thebird in multiple doses wherein an initial immunization is followed bybooster immunizations.

The composition can be administered to an animal at any time before theanimal ceases to ovulate or produce sperm due to disease or age. Thepreferred age at which the composition of the present invention isadministered to an animal depends upon the species of the animalinvolved, the mating season (if any) of an animal, and upon the purposeof the administration of the composition.

For example, where the composition is administered to accelerate theonset of egg lay or sperm production, the composition of the presentinvention is to be administered to a bird before the bird reaches egglay or puberty. As stated above, the preferred age at which thecomposition of the present invention is first administered to an animaldepends upon the species of the animal involved, the mating season (ifany) of an animal, upon the size of the bird, and upon the identity ofthe components (inhibin and carrier protein) in the composition.

As another example, where the composition is administered to enhanceproduction performance of agricultural animals which have breedingseasons, the preferred time of administering the composition is prior tothe start of the breeding season. In contrast, where the composition isto be administered to a mature animal which has a suppressed eggproduction rate or a suppressed sperm production rate, then thecomposition would be administered at the time that the suppression isrecognized as problematic.

With respect to an animal having a breeding season, although theheterologous protein of the present invention can be administered to abird such as a ratite at any age, immunizing the bird during the sixmonths prior to the bird's first breeding season is preferable. It isunderstood by those of ordinary skill in the art that average femalebirds initiate egg lay during the first breeding season. It is even morepreferable to immunize the bird approximately six months prior to thebird's first breeding season, and then to administer boosterimmunizations at one month intervals prior to the bird's first breedingseason. It is most preferable to immunize the bird approximately sixmonths prior to the bird's first breeding season, and then to administerbooster immunizations at one month intervals for six months.

For example, for best results in increasing the egg production of afemale ostrich, a primary immunization is administered to the ostrichapproximately 6 months before its first breeding season, and thenbooster immunizations are administered at one month intervals for sixmonths. The primary immunization comprises between approximately 0.5 to4.5 mg of the heterologous protein of the present invention. The boosterimmunizations comprise between approximately 0.30 to 3.0 mg of theheterologous protein of the present invention. Preferably, the primaryimmunization comprises between approximately 1.5 to 3.0 mg of theheterologous protein of the present invention. The booster immunizationscomprise between approximately 0.75 to 1.5 mg of the heterologousprotein of the present invention. It is also preferable that theheterologous protein is emulsified in Freund's Complete Adjuvant (SigmaChemical Co., St. Louis, Mo.) in the primary immunization, and that theheterologous protein is emulsified in Freud's Incomplete Adjuvant(Sigma) in the booster immunizations. Even more preferably, theheterologous protein composition is injected subcutaneously. Mostpreferably, the heterologous protein composition is injectedsubcutaneously at three sites along the upper thigh region of theostrich.

The amount administered to a bird of the heterologous protein of thepresent invention varies according to the species of the bird, the ageand weight of the bird, when the protein is administered in relation tothe breeding season (if the bird has a breeding season), and how manytimes the protein is to be administered. Also, the commencement of theadministration schedule, or treatment schedule, varies according to thespecies of the bird, the average age of puberty of that species of thebird, the family history of the bird (with respect to the family'shistory of age at puberty), the time of year the bird was hatched, thenutritional plane of the bird (highly fed birds come into puberty beforethose that are undernourished), the general health of the bird at thattime of commencement, immunological competence of the bird, the longterm health history of the bird, the presence of extreme weatherconditions (prolonged excessive inclement weather such as rain, heat, orwindiness that the bird is not accustomed to), housing conditions(overcrowding), and a lack of exercise.

One of ordinary skill in the art, in view of the teachings of thepresent invention, would be able to determine by routine testing theamount of heterologous protein that will be necessary to elicit animmunological response to the protein by the bird.

Another example of the method for enhancing production performance is asfollows. An immunologically effective amount of a conjugatedheterologous protein composition is administered to a mammal such thatan immunological response occurs in the mammal which is directed againstthe heterologous protein. The heterologous protein is preferablycomprised of mammalian inhibin conjugated to maltose binding protein.Another preferred conjugated heterologous protein is comprised of avianor reptilian inhibin, and maltose binding protein.

For example, the following is a brief summary of the method of thepresent invention for enhancing production performance in Japanese Quailas is fully discussed in Example 8. The average age at puberty for anuntreated quail is approximately six to eight weeks. The following wouldbe the treatment schedule for Japanese quail having an approximate bodyweight range of 0.1 to 0.25 pounds: primary (first) injection of 0.75 mgof the heterologous protein of the present invention on its 25th day ofage; and boosters of 0.375 mg on the 32nd, 39th, 46th, 53rd, 60th, and90th day of age, followed by boosters every 35 days thereafter for threeadditional challenges (i.e., at 95, 130, and 165 days-of-age.).

More particularly, at 25 days-of-age, 50 female quail were randomly andequally assigned to one of two injection groups (25 birds per group) asfollows: (1) MBP-cINA₅₂₁ in Freund's adjuvant ("MBP-cINA₅₂₁ /FRN"), or(2) Freund's (adjuvant control; "FRN"). Birds immunized against inhibin(Group 1) were given approximately 0.75 mg MBP-cINA₅₂₁ per bird in theappropriate control vehicle. Equivalent vehicular injection volumes (0.2mL) of FRN were used in Group 2. All injections were givensubcutaneously using tuberculin syringes fitted with 25 gauge needles.As discussed above, booster inhibin immunizations of approximately 0.375mg MBP-cINA₅₂₁ per bird, or appropriate control challenges, weresubsequently administered and the birds were observed for a total of 20weeks

Beginning at 41 days-of age, which is considered to be day 1 of the egglay cycle, daily hen-day egg production ("HDEP") and mortality ("MORT")measures were recorded for 20 consecutive weeks. In addition, averageage at first egg lay ("FIRST") and age at which hens reached 50% eggproduction ("FIFTY") were calculated for each of the treatment groups.As is more fully discussed in Example 8, HDEP, MORT, FIRST, and FIFTYdata were subjected to analyses of variance.

Inhibin immunoneutralization clearly accelerated puberty in the quailhens. As shown in Table 2, the average age of FIRST egg lay wasdecreased (P<0.0088) by nearly six days in inhibin-treated hens.Likewise, as shown in Table 3, the age to FIFTY egg production wasmarkedly reduced (12 days; P<0.01) in inhibin-treated hens.

A positive effect of inhibin treatment on intensity of egg lay was alsoextant, most notably at the beginning and at the end of the layingcycle, as shown in FIG. 2. For example, significantly greater (P<0.05)mean HDEP rates were observed in hens treated with MBP-cINA₅₂₁ /FRN whencompared to the FRN controls during Weeks 1 (16.5 vs 2.6%), 2 (50.0 vs28.6%), and 4 (96.6 vs 79.7%) and again during Weeks 15 (98.8 vs 86.9%),16 (96.9 vs 86.3%), 18 (85.7 vs 66.1%), and 20 (96.8% vs 73.8%). TotalHDEP rate, inclusive of all 20 weeks of lay, for inhibin-treated henswas 83.5% as compared to 75.4% for the controls (P<0.14).

Besides accelerating puberty, prolonging egg lay, and enhancing theoverall intensity of lay, inhibin-treatment decreased the time needed toreach peak egg lay by approximately 3 weeks. Referring now to FIG. 2,compare MBP-cINA₅₂₁ /FRN which is at 96.6% HDEP by Week 4 versus FRNwhich is at 96.6% HDEP by Week 7. Although differences in peak HDEPvalues were not statistically evaluated, the treatment differences inmean age at which hens reached 50% HDEP levels (FIFTY) reflect peakperformance.

Mortality was not a factor in this study as only eight birds have died(three controls, five treated). The mortality of 16% is within expectedlimits for quail that have reached 180 days-of-age.

Most timed biological responses to treatments are studied for effects ononset, magnitude, and duration of response. Herein, the data representwhat most would consider to be a full cycle of lay in Japanese quail(i.e., 20 weeks post-initiation of puberty or egg lay). Therefore, thefollowing comments on the effects of inhibin immunoneutralization on theonset, magnitude and duration of egg lay in this species are justified.

The data support the conclusion that onset of puberty was accelerated inthe inhibin immunoneutralized group. This was evidenced in the markedtreatment differences noted in both the FIRST and FIFTY variables and inthe differences observed during the initial weeks of HDEP data.

The acceleration of puberty coupled with the increased persistency ofegg lay in the inhibin-challenged birds contributed to an increase inoverall HDEP that was marked (8.1%). For example, on a per-hen basis,inhibin treatment essentially translated into a daily gain ofapproximately 0.081 eggs for every day of the laying cycle that a henremained viable (ie., capable of laying an egg). This means thatapproximately 11 more eggs were obtained for each hen housed during the20 week period examined (0.081 eggs/hen×140 days-of-lay =11.34 eggs perhen per laying cycle).

Similar results in chickens and turkeys, as found in Coturnix, will havesubstantial strategic relevance to the poultry industry. It should benoted that Japanese quail have been selected for intensity of egg lay,and that egg laying potential is considered to be even greater inCoturnix than in chicken hens (Single Comb White Leghorns) commerciallyreared for the single purpose of production of table eggs. Therefore,intensification of egg lay by inhibin vaccination in chickens which havenot been selected for egg production but for meat production, e.g.,broiler breeders raised for the consumption of their flesh, may be evengreater.

Accordingly, the above data shows that the inhibin composition of thepresent invention enhances production performance as it accelerates theonset of puberty, increases egg lay intensity, and accelerates the onsetof maximum egg lay in Japanese Quail. Since Japanese Quail are anacceptable animal model for chickens with respect to their reproductivesystems, the above data indicates that the method of the presentinvention will also accelerate the onset of egg lay in chickens.Accordingly, the method of the present invention will result in an eggproducer being able to produce more eggs with lower feed costs.

The above data also shows that the inhibin composition of the presentinvention enhanced production performance as it minimized the adverseaffects of elevated temperatures of the egg lay rate of the JapaneseQuail. More particularly, in the eighteenth week of the study describedin Example 8, the Quail were inadvertently exposed to elevatedtemperatures. As can be seen in FIG. 2, the birds in Group 1 (treatedwith MBP-cINA_(521/) FRN) sustained a drop in egg lay rate ofapproximately 5%. In contrast, the birds in Group 2 (control: FRN)sustained a drop in egg lay rate of approximately 26%. Accordingly, themethod of the present invention of enhancing production performanceameliorates the negative impact on egg lay rates of poultry exposed toadverse egg laying conditions. This aspect of the present invention issignificant as poultry are often raised in open, uncontrolledenvironments. Accordingly, poultry stocks are often exposed to adverseconditions such as elevated temperatures, and other extreme weatherconditions that they are not acclimated to, which thereby decrease egglay rates in the poultry industry.

The following is a brief summary of the method of the present inventionfor enhancing production performance in ostriches as is discussed inExample 9. The average age at puberty for untreated ostriches is betweenapproximately 28 and 32 months. The following would be the treatmentschedule for ostriches having an approximate body weight range of 150 to300 pounds: primary (first) injection of 5.0 mg of the heterologousprotein of the present invention on its 26th month of age; and boostersof 2.5 mg on the 27th, 28th, 30th, 32nd, 34th, and 36th month of age.

The following is a brief summary of the method of the present inventionfor enhancing production performance in emu as is discussed in Example10. The average age at puberty for untreated emu is approximately 20months. The following would be the treatment schedule for emu having anapproximate body weight range of 50 to 90 pounds: primary (first)injection of 3.0 mg of the heterologous protein of the present inventionon its 18th month of age; and boosters of 1.5 mg on the 19th, 20th,22nd, 24th, 26th, and 30th month of age.

The following is a brief summary of the method of the present inventionfor enhancing production performance in chickens as is discussed inExample 11. The average age at puberty for an untreated chicken isapproximately 20 weeks. The following would be the treatment schedulefor a chicken having an approximate body weight range of 2.0 to 3.5pounds: primary (first) injection of 1.5 mg of the heterologous proteinof the present invention on its 15th week of age; and boosters of 0.75mg on the 17th, 20th, 24th, 30th, 40th, and 50th week of age.

The following is a brief summary of the method of the present inventionfor enhancing production performance in turkeys as is discussed inExample 12. The average age at puberty for an untreated turkey isapproximately 30 weeks. The following would be the treatment schedulefor a turkey having an approximate body weight range of 9.0 to 12pounds: primary (first) injection of 2.0 mg of the heterologous proteinof the present invention on its 28th week of age; and boosters of 1.0 mgon the 29th, 30th, 34th, 38th, 46th, and 54th week of age.

The following is a brief summary of the method of the present inventionfor enhancing production performance in parrots as is discussed inExample 13. The average age at puberty for an untreated parrot isapproximately 30 months. The following would be the treatment schedulefor a parrot having an approximate body weight range of 0.5 to 1.25pounds: primary (first) injection of 0.75 mg of the heterologous proteinof the present invention on its 28th month of age; and boosters of 0.375mg on the 29th, 30th, 32nd, 34th, 36th, and 38th month of age.

As discussed above, the method of the present invention enhancedproduction performance by accelerating the onset of puberty in theanimal that the composition of the present invention was administeredto. The term "accelerates" with respect to the onset of egg lay denotesthat egg lay of a treated bird commences at least about 3% earlier thanegg lay would ordinarily commence in an untreated bird. Preferably, egglay commences at least about 5% earlier, and more preferably commencesat least about 7% earlier. Even more preferably, egg lay commences atleast about 10% earlier, and most preferably commences at least about13% earlier than egg lay would ordinarily commence in an untreated bird.

Also, as discussed above, the method of the present invention enhancedproduction performance by increasing egg or sperm production intensityin animals. The term "increases" with respect to egg production denotesthat egg production of a treated bird increases at least about 3% withrespect to the amount of egg production in an untreated bird.Preferably, egg production increases at least about 7%, and morepreferably increases at least about 12%.

Further, as discussed above, the method of the present inventionenhances production performance by accelerating the onset of maximum eggproduction in an animal. The term "accelerates" with respect to theonset of maximum egg lay denotes that maximum egg lay of a treated birdcommences at least about 3% earlier than egg lay would ordinarilycommence in an untreated bird. Preferably, maximum egg lay commences atleast about 5% earlier, and more preferably commences at least about 7%earlier. Even more preferably, maximum egg lay commences at least about10% earlier, and most preferably commences at least about 13% earlierthan maximum egg lay would ordinarily commence in an untreated bird.

Surprisingly, the composition of the present invention can also be usedto increase the lifetime total egg lay of birds. The term "increase"with respect to total lifetime egg lay denotes that the total lifetimeegg lay of a treated bird increases at least about 3% with respect tothe total lifetime egg lay of an untreated bird. Preferably, totallifetime egg lay increases at least about 7%, and more preferablyincreases at least about 12%. Most preferably, total lifetime egg layincreases at least about 15%.

Unexpectedly, the composition of the present invention can also be usedto decrease or eliminate the need to molt a female bird, e.g., toprolong egg laying persistency by providing for a second cycle of lay.More particularly, if the composition described above is continuallyadministered to the female bird, as disclosed in the method above, therate of egg lay of the bird, in comparison to if the bird was nottreated with the composition of the present invention, would remain highenough so that the bird would not need to be molted to improve its rateof egg lay. It is a common practice in the art to molt a female bird,such as chicken hens (Single Comb White Leghorns, table egg producers),when its egg lay production declines such that the economic cost ofmaintaining the bird outweighs the economic benefit yielded by the eggsproduced. To "molt" a chicken hen, the bird undergoes a period offasting of approximately four to fourteen days until it beings to molt,e.g., lose its feathers. During the molting period, the bird stopslaying eggs. After the bird is placed back onto normal levels of feed,egg production recommences after a period of time. The entire moltingperiod is approximately two months from the beginning of the fast periodto the onset of the next egg-lay cycle. In effect, the egg productionrate of the bird is rejuvenated. However, after molting a chicken, itsrate of egg-lay in the next cycle does not equal the egg productionduring the first (pre-molt) egg-lay cycle. M. North and D. Bell,Commercial Chicken Production Manual, fourth edition, Chapter 19,Published by Van Norstrand Reinhold of New York.

For example, chickens reach egg lay at approximately 20 weeks, andproduce an economically significant number of eggs for approximately 40to 50 weeks. At the peak of egg lay, chickens produce eight to nine eggsevery ten days. However, after approximately 50 weeks of egg lay, therate of egg production decreases to approximately 60% of peak egg lay.At this point, the cost of the feed for the chicken is greater than thevalue of the eggs its produces. It is common practice to molt thechicken at this point, so that when the chicken recommences egg lay, itsrate of egg lay is increased. By "prolonging the persistence of egg lay"with reference to chickens and quail, among other birds, it is meantthat egg lay will be prolonged for approximately one to four weeks.

Therefore, the composition of the present invention, as it maintains therate of egg lay at a higher level than if the bird were not treated withthe composition, reduces or eliminates the need to molt a bird. Thereduction or elimination of the need to molt a bird results insignificant savings. More particularly, during the period that a bird ismolted, and prior to that time, the bird has been unproductive withrespect to its feed cost before it is molted, and then it isunproductive for a period of time after feeding recommences. Maintainingthe rate of egg lay at an enhanced level therefore eliminates or reducesthese unproductive phases of the bird, thereby reducing the producer'scosts and increasing the producer's profits. Maintaining the rate of egglay at an enhanced level further enhances egg producer's profits as therate of egg-lay after molting does not equal the rate of egg-lay in thefirst cycle of egg lay as discussed above.

Briefly described, the rate of egg lay of birds would be enhanced,thereby avoiding the need to molt the bird, by administering aneffective amount of the heterologous protein of the present invention toinduce an immunological response thereto, and thereafter administeringan effective amount of the heterologous protein (boosters) to maintain ahigher than normal rate of egg lay.

Accordingly, the method of the present invention enhances productionperformance in female animals which produce inhibin, such as mammals,reptiles, and birds such as ratites. More particularly, this methodenhances production performance in female ratites such as ostriches,emus, and rhea, and in chickens. Unexpectedly, the method of the presentinvention increases the onset of puberty or first egg lay in animals.Also, the method of the present invention accelerates the onset ofmaximum egg lay in an animal. Further, the method of the presentinvention increases the number of eggs laid by an animal. Further still,the method of the present invention prolongs the persistence of maximumegg lay in animals. Still further, the method increases the lifetimetotal egg lay of an animal. In avians, the method of the presentinvention also improves the feed conversion ratio of the bird. Also, themethod of the present invention unexpectedly reduces or eliminates theeffect of adverse laying conditions on egg lay rates of animals exposedto such conditions. Such adverse conditions include elevatedtemperatures, overcrowding, poor nutrition, and noise.

Although not wanting to be limited by the following, it is theorizedthat the method of the present invention of enhancing productionperformance in animals provides a greater increase in egg production inspecies that have not been genetically selected for the trait ofprolific egg laying. This is particularly true for certain avians. Forexample, egg-type chickens have been genetically selected for maximumproduction performance since the late 1920s. (See, for example, Jull,M.A, 1932, Poultry Breeding, John Wiley & Sons.) In terms of the shortlife span of a chicken, a great deal of selection for this traitoccurred over the period of time from approximately 1928 to the present.In contrast, ratites and psittaciformes, most other exotic birds, and toa lesser extent meat-type chickens (broilers) have not been geneticallyselected for the trait of prolific egg laying. Also, birds that areendangered have also not been genetically selected for the trait ofprolific egg laying. Accordingly, as egg-type chickens are alreadygenetically excellent egg layers, the amount of improvement that can beseen with the method of the present invention is limited in comparisonto birds that are genetically poor to medium egg layers. Therefore, amuch greater amount of improvement in the production performance is seenwith the method of the present invention with birds that have not beengenetically selected for prolific egg laying, such as, ratites,psittaformes, other exotic birds, endangered birds, turkeys, andmeat-type chickens.

The immunization of an animal with the heterologous protein of thepresent invention induces the animal to produce antibodies selectivelydirected against the heterologous protein. Preferably, the immunizationalso induces the animal to produce antibodies selectively directedagainst endogenous inhibin. The production of such antibodies by a birdreduces the time to the onset of puberty or egg lay. The production ofsuch antibodies by the animal also enhances the animal's egg productioncapability or sperm production capability as the antibodies neutralizethe biological activity of inhibin in the animal's blood stream.

Not wanting to be bound by the following theory, it is believed that theα-subunit of inhibin binds to FSH receptors and therefore competitivelyinhibits the binding of FSH with such receptor sites. Reducing thelevels of inhibin that may bind with receptor sites, therefore increasesthe biological effect of the FSH in the animal as there is reducedcompetition for the FSH receptor sites. It is thought that theantibodies neutralize the inhibin by interacting with the circulatinginhibin, thereby stearically interfering with the binding of theinteracted inhibin to the FSH receptor sites.

Unexpectedly, the method of the present invention also improvesproduction performance in male animals which produce inhibin, such asmammals, reptiles, and birds. More particularly, the method of thepresent invention increases testosterone levels in male animals.Similarly, the method of the present invention increases the onset ofpuberty or sperm production in male animals. Also, the method of thepresent invention accelerates the onset of maximum sperm production in amale animal. Further, the method of the present invention increases theintensity of sperm production (sperm count) by a male animal. Furtherstill, the method of the present invention prolongs the persistence ofmaximum sperm production in animals. Also, the method of the presentinvention increases ejaculate volume in male animals. Further, themethod improves sperm viability in animals. Still further, the methodunexpectedly reduces or eliminates the effect of adverse conditions onsperm production of animals exposed to such conditions. Such adverseconditions include elevated temperatures, overcrowding, poor nutrition,and noise. The method of the present invention also unexpectedlyincreases the libido, and therefore, the reproductive potential, of amale bird.

Another unexpected and surprising aspect of the present invention isthat the composition of the present invention also produces more eggshaving a decreased cholesterol content in comparison to the eggs laid byuntreated birds. More particularly, if the composition described aboveis administered to a female bird, as disclosed in the method above, thecholesterol content of the eggs laid by the bird will be reduced for alonger period of time in comparison to if the bird was not treated withthe composition of the present invention. Therefore, the composition ofthe present invention increases or produces a greater number of lowcholesterol eggs.

The terms "increase" or "greater number of" denotes that the number oflow cholesterol eggs produced by a treated bird increases at least about2% with respect to the number of low cholesterol eggs produced by anuntreated bird. Preferably, the number of low cholesterol eggs producedincreases at least about 5%, and more preferably increases at leastabout 10%. It is to be understood that a "treated" bird is a bird towhich the heterologous protein of the present invention has beenadministered. The term "lower cholesterol" or "low cholesterol" denotesthat the cholesterol content of an egg is lower than the averagecholesterol content of eggs produced by that species of bird during thelifetime of such a bird by at least about 10%. Preferably, thecholesterol content of a low cholesterol egg is lower than the averageby at least about 20%. More preferably, the cholesterol content of a lowcholesterol egg is lower than the average by at least about 30%.

It is known that in chickens, the first five to six eggs that are laidby a female bird (hen) after it reaches puberty are lower in cholesterolthan the eggs it later produces. The composition of the presentinvention induces the female bird to lay eggs having a lower cholesterolcontent for a longer period of time. Due to the health consequenceslinked to high cholesterol levels in the blood, there remains a need forlow cholesterol egg products. Accordingly, the composition of thepresent invention provides the producer of eggs with a greater number ofa highly sought after type of egg.

Gene Therapy using the Fusion Gene Product

The present invention also relates to a method of enhancing theproduction performance of animals, by administering to the animal afusion gene product comprising a gene encoded for the expression ofalpha-subunit inhibin protein, or a fragment thereof, and a gene encodedfor the expression of a carrier protein. The fusion gene product of thepresent invention may be administered directly to the animal, or it maybe administered in a vector, or in a cell containing a vector having thefusion gene product therein.

Various methods of transferring or delivering DNA to cells forexpression of the gene product protein, otherwise referred to as genetherapy, are disclosed in Gene Transfer into Mammalian Somatic Cells invivo, N. Yang, Crit. Rev. Biotechn. 12(4): 335-356 (1992), which ishereby incorporated by reference. Gene therapy encompasses incorporationof DNA sequences into somatic cells or germ line cells for use in eitherex vivo or in vivo therapy. Gene therapy functions to replace genes, andaugment normal or abnormal gene function.

Strategies for gene therapy include therapeutic strategies such asidentifying a defective gene and then adding a functional gene to eitherreplace the function of the defective gene or to augment a slightlyfunctional gene; or prophylactic strategies, such as adding a gene forthe product protein. As an example of a prophylactic strategy, a fusedgene product which encodes for inhibin, or a fragment thereof, and acarrier protein may be placed in an animal thereby secondarily reducingthe levels of inhibin in the animal due to the immune response.

Any protocol for transfer of the fused gene product of the presentinvention is contemplated as part of the present invention. Transfectionof promoter sequences, other than one normally found specificallyassociated with inhibin, or other sequences which would decreaseproduction of inhibin protein are also envisioned as methods of genetherapy. An example of this technology is found in TranskaryoticTherapies, Inc., of Cambridge, Massachusetts, using homologousrecombination to insert a "genetic switch" that turns on anerythropoietin gene in cells. See Genetic Engineering News, April 15,1994.

Gene transfer methods for gene therapy fall into three broadcategories-physical (e.g., eiectroporation, direct gene transfer andparticle bombardment), chemical (lipid-based carriers, or othernon-viral vectors) and biological (virus-derived vector and receptoruptake). For example, non-viral vectors may be used which includeliposomes coated with DNA. Such liposome/DNA complexes may be directlyinjected intravenously into the animal. It is believed that theliposome/DNA complexes are concentrated in the liver where they deliverthe DNA to macrophages and Kupffer cells. These cells are long lived andthus provide long term expression of the delivered DNA. Additionally,vectors or the "naked" DNA of the gene may be directly injected into thedesired organ, tissue or tumor for targeted delivery of the therapeuticDNA.

Gene therapy methodologies can also be described by delivery site.Fundamental ways to deliver genes include ex vivo gene transfer, in vivogene transfer, and in vitro gene transfer. In ex vivo gene transfer,cells are taken from the animal and grown in cell culture. The DNA istransfected into the cells, the transfected cells are expanded in numberand then reimplanted in the animal. In in vitro gene transfer, thetransformed cells are cells growing in culture, such as tissue culturecells, and not particular cells from a particular animal. These"laboratory cells" are transfected, the transfected cells are selectedand expanded for either implantation into an animal or for other uses.

In vivo gene transfer involves introducing the DNA into the cells of theanimal when the cells are within the animal. Methods include usingvirally mediated gene transfer using a noninfectious virus to deliverthe gene in the animal or injecting naked DNA into a site in the animaland the DNA is taken up by a percentage of cells in which the geneproduct protein is expressed. Additionally, the other methods describedherein, such as use of a "gene gun," may be used for in vitro insertionof inhibin DNA or inhibin regulatory sequences.

Chemical methods of gene therapy may involve a lipid based compound, notnecessarily a liposome, to ferry the DNA across the cell membrane.Lipofectins or cytofectins, lipid-based positive ions that bind tonegatively charged DNA, make a complex that can cross the cell membraneand provide the DNA into the interior of the cell. Another chemicalmethod uses receptor-based endocytosis, which involves binding aspecific ligand to a cell surface receptor and enveloping andtransporting it across the cell membrane. The ligand binds to the DNAand the whole complex is transported into the cell. The ligand genecomplex is injected into the blood stream and then target cells thathave the receptor will specifically bind the ligand and transport theligand-DNA complex into the cell.

Many gene therapy methodologies employ viral vectors to insert genesinto cells. For example, altered retrovirus vectors have been used in exvivo methods to introduce genes into peripheral and tumor-infiltratinglymphocytes, hepatocytes, epidermal cells, myocytes, or other somaticcells. These altered cells are then introduced into the animal toprovide the gene product from the inserted DNA.

Viral vectors have also been used to insert genes into cells using invivo protocols. To direct tissue-specific expression of foreign genes,cis-acting regulatory elements or promoters that are known to be tissuespecific can be used. Alternatively, this can be achieved using in situdelivery of DNA or viral vectors to specific anatomical sites in vivo.For example, gene transfer to blood vessels in vivo was achieved byimplanting in vitro transduced endothelial cells in chosen sites onarterial walls. The virus infected surrounding cells which alsoexpressed the gene product. A viral vector can be delivered directly tothe in vivo site, by a catheter for example, thus allowing only certainareas to be infected by the virus, and providing long-term, sitespecific gene expression. In vivo gene transfer using retrovirus vectorshas also been demonstrated in mammary tissue and hepatic tissue byinjection of the altered virus into blood vessels leading to the organs.

Viral vectors that have been used for gene therapy protocols include butare not limited to, retroviruses, other RNA viruses such as poliovirusor Sindbis virus , adenovirus, adeno-associated virus, herpes viruses,SV 40, vaccinia and other DNA viruses. Replication-defective murineretroviral vectors are the most widely utilized gene transfer vectors.Murine leukemia retroviruses are composed of a single strand RNAcomplexed with a nuclear core protein and polymerase (pol) enzymes,encased by a protein core (gag) and surrounded by a glycoproteinenvelope (env) that determines host range. The genomic structure ofretroviruses include the gag, pol, and env genes enclosed by the 5' and3' long terminal repeats (LTR). Retroviral vector systems exploit thefact that a minimal vector containing the 5' and 3' LTRs and thepackaging signal are sufficient to allow vector packaging, infection andintegration into target cells providing that the viral structuralproteins are supplied in trans in the packaging cell line. Fundamentaladvantages of retroviral vectors for gene transfer include efficientinfection and gene expression in most cell types, precise single copyvector integration into target cell chromosomal DNA, and ease ofmanipulation of the retroviral genome.

The adenovirus is composed of linear, double 5 stranded DNA complexedwith core proteins and surrounded with capsid proteins. Advances inmolecular virology have led to the ability to exploit the biology ofthese organisms in order to create vectors capable of transducing novelgenetic sequences into target cells in vivo. Adenoviral-based vectorswill express gene product peptides at high levels. Adenoviral vectorshave high efficiencies of infectivity, even with low titers of virus.Additionally, the virus is fully infective as a cell free virion soinjection of producer cell lines are not necessary. Another potentialadvantage to adenoviral vectors is the ability to achieve long termexpression of heterologous genes in vivo.

Mechanical methods of DNA delivery include fusogenic lipid vesicles suchas liposomes or other vesicles for membrane fusion, lipid particles ofDNA incorporating cationic lipid such as lipofectin, polylysine-mediatedtransfer of DNA, direct injection of DNA, such as microinjection of DNAinto germ or somatic cells, pneumatically delivered DNA-coatedparticles, such as the gold particles used in a "gene gun," andinorganic chemical approaches such as calcium phosphate transfection.Another method, ligand-mediated gene therapy, involves complexing theDNA with specific ligands to form ligand-DNA conjugates, to direct theDNA to a specific cell or tissue.

It has been found that injecting plasmid DNA into muscle cells yieldshigh percentage of the cells which are transfected and have sustainedexpression of marker genes. The DNA of the plasmid may or may notintegrate into the genome of the cells. Non-integration of thetransfected DNA would allow the transfection and expression of geneproduct proteins in terminally differentiated, non-proliferative tissuesfor a prolonged period of time without fear of mutational insertions,deletions, or alterations in the cellular or mitochondrial genome.Long-term, but not necessarily permanent, transfer of therapeutic genesinto specific cells may provide treatments for genetic diseases or forprophylactic use. The DNA could be reinjected periodically to maintainthe gene product level without mutations occurring in the genomes of therecipient cells. Non-integration of exogenous DNAs may allow for thepresence of several different exogenous DNA constructs within one cellwith all of the constructs expressing various gene products.

Particle-mediated gene transfer methods were first used in transformingplant tissue. With a particle bombardment device, or "gene gun," amotive force is generated to accelerate DNA-coated high densityparticles (such as gold or tungsten) to a high velocity that allowspenetration of the target organs, tissues or cells. Particle bombardmentcan be used in in vitro systems, or with ex vivo or in vivo techniquesto introduce DNA into cells, tissues or organs.

Electroporation for gene transfer uses an electrical current to makecells or tissues susceptible to electroporation-mediated gene transfer.A brief electric impulse with a given field strength is used to increasethe permeability of a membrane in such a way that DNA molecules canpenetrate into the cells. This technique can be used in in vitrosystems, or with ex vivo or in vivo techniques to introduce DNA intocells, tissues or organs.

Carrier mediated gene transfer in vivo can be used to transfect foreignDNA into cells. The carrier-DNA complex can be conveniently introducedinto body fluids or the bloodstream and then site specifically directedto the target organ or tissue in the body. Both liposomes andpolycations, such as polylysine, lipofectins or cytofectins, can beused. Liposomes can be developed which are cell specific or organspecific and thus the foreign DNA carried by the liposome will be takenup by target cells. Injection of immunoliposomes that are targeted to aspecific receptor on certain cells can be used as a convenient method ofinserting the DNA into the cells bearing the receptor. Another carriersystem that has been used is the asialoglycoportein/polylysine conjugatesystem for carrying DNA to hepatocytes for in vivo gene transfer.

The transfected DNA may also be complexed with other kinds of carriersso that the DNA is carried to the recipient cell and then resides in thecytoplasm or in the nucleoplasm. DNA can be coupled to carrier nuclearproteins in specifically engineered vesicle complexes and carrieddirectly into the nucleus.

Gene regulation of inhibin may be accomplished by administeringcompounds that bind to the inhibin gene, or control regions associatedwith the inhibin gene, or its corresponding RNA transcript to modify therate of transcription or translation. Additionally, cells transfectedwith a DNA sequence encoding inhibin, or a fragment thereof, and acarrier protein may be administered to an animal to provide an in vivosource of the heterologous protein of the present invention. Forexample, cells may be transfected with a vector containing the fusiongene product of the present invention, encoding inhibin, or a fragmentthereof, and a carrier protein.

The term "vector" as used herein means a carrier that can contain orassociate with specific nucleic acid sequences, which functions totransport the specific nucleic acid sequences into a cell. Examples ofvectors include plasmids and infective microorganisms such as viruses,or non-viral vectors such as ligand-DNA conjugates, liposomes, lipid-DNAcomplexes. It may be desirable that a recombinant DNA moleculecomprising the fused gene product of the present invention isoperatively linked to an expression control sequence to form anexpression vector capable of expressing the heterologous protein of thepresent invention. The transfected cells may be cells derived from theanimal's normal tissue, the animal's diseased tissue, or may benon-animal cells.

For example, cells removed from an animal can be transfected with avector capable of expressing the heterologous protein of the presentinvention, and re-introduced into the animal. The transfected cells thenproduce the heterologous protein of the present invention thus inducingan immunological response to the inhibin. Cells may also be transfectedby non-vector, or physical or chemical methods known in the art such aselectroporation, ionoporation, or via a "gene gun." Additionally, thefused gene product of the present invention may be directly injected,without the aid of a carrier, into an animal. In particular, the fusedgene product of the present invention may be injected into skin, muscleor blood.

The gene therapy protocol for transfecting inhibin, or a fragmentthereof, into an animal may either be through integration of the fusedgene product into the genome of the cells, into minichromosomes, or as aseparate replicating or non-replicating DNA construct in the cytoplasmor nucleoplasm of the cell. Heterologous protein expression may continuefor a long-period of time, or the fused gene product of the presentinvention may be reinjected periodically to maintain a desired level ofthe heterologous protein in the cell, the tissue, or organ, or adetermined blood level.

The fused gene product of the present invention can be administered to abird by any means known in the art. For example, the composition can beadministered subcutaneously, intraperitonealy, intradermally,intravascularly, or intramuscularly. Preferably, the composition isinjected subcutaneously. Another preferable administration isintravascular infusion near the preferred site of therapy. Thecomposition can be administered to the bird in one or more doses.Preferably, the composition is administered to the bird in multipledoses wherein an initial immunization is followed by boosterimmunizations. The preferred amount of the fused gene product to beadministered is between 50 and 300 micrograms per kilogram of bodyweight. Preferably, the fused gene product is administered in a carrier,such as a buffer or Freund's adjuvant.

The methods of the present invention for enhancing productionperformance in birds will greatly accelerate the growth of thepopulation and therefore the market for ratites such as ostrich and emuas their suboptimal egg laying rates will be increased by the presentmethod. The method of the present invention will also satisfy theexpanding need for poultry such as domestic chickens, and their eggs.

The utility of the method of the present invention for enhancingproduction performance is not limited to enhancing productionperformance in birds. The present method for enhancing productionperformance can be used in many animals. As stated above, the method ofthe present invention is used to enhance production performance of anyanimal that produces inhibin, including, but not limited to, mostanimals that are raised agriculturally, such as pigs, cows, sheep,turkeys, quail, ducks, geese, turtles, fish, and chickens; in furbearing animals such as mink, fox, otter, ferret, rabbits, and raccoons;rodents for laboratory testing such as mice, rats, hamsters, guinea pigsand gerbils; for animals whose hides are used for decorative purposessuch as alligators and snakes; exotic or endangered species; animalsused for racing, entertainment, or showing (competitions) such ashorses, dogs, cats, zoo animals, and circus animals; and humans.Additional avians that the method of the present invention enhancesproduction performance thereof include ratites, psittaciformes,falconiformes, piciformes, strigiformes, passeriformes, coraciformes,ralliformes, cuculiformes, columbiformes, galliformes, anseriformes, andherodiones. More particularly, the method of the present invention maybe used to enhance production performance of an ostrich, emu, rhea,kiwi, cassowary, parrot, parakeet, makaw, falcon, eagle, hawk, pigeon,cockatoo, song bird, jay bird, blackbird, finch, warbler, canary,toucan, mynah, or sparrow.

Qualitative or Quantitative Methods of the Present Invention

Another aspect of the present invention is a method of producingantibodies directed against the heterologous protein of the presentinvention. Generally, the method of producing antibodies directedagainst the heterologous protein comprises the steps of: administeringan effective amount of a heterologous protein comprising inhibinprotein, or a fragment thereof, and a carrier protein, to an animal suchthat an immunological response occurs in the animal against theheterologous protein; removing a blood sample from the animal; and thenisolating any antibodies directed against inhibin from the serum of theblood sample. Preferably, the antibodies are isolated from the serum ofthe blood sample by passing the serum through a column containingeffective amounts of the carrier protein to separate the antibodies fromthe serum. Alternatively, the column will contain the heterologousprotein of the present invention. In another technique, the antibodiesare isolated by first passing it through a column containing the carrierprotein, and then passing it through a column containing theheterologous protein of the present invention. The techniques used toproduce and purify antibodies directed against the heterologous proteinof the present invention are well known to one of ordinary skill in theart.

It is also to be understood that the heterologous protein of the presentinvention can be administered to any animal dependent upon the type ofantibodies that are desired. Further, it is to be understood that theinhibin can be exogenous or endogenous. Therefore, the type of inhibinin the heterologous protein of the present invention, and the species ofanimal the composition is administered to is determined by what type ofantibody is desired. For example, heterologous protein comprisingchicken inhibin and maltose binding protein can be administered to anostrich to produce ostrich anti-chicken inhibin antibodies. Also,heterologous protein comprising ostrich inhibin and maltose bindingprotein can be administered to an ostrich to produce ostrichanti-ostrich inhibin antibodies.

The present invention is also directed to a rapid, simple, reliable,cost-effective method for determining whether an animal is hormonallypre-dispositioned to be a high-level or low-level egg producer. Moreparticularly, the present invention also relates to a method ofdetermining the amount of inhibin produced by an animal, which thereforeallows the determination of the egg production capability of the animal.Briefly described, the method of determining the quantity of inhibin inthe blood of an animal, comprises the steps of: withdrawing a bloodsample from an animal; contacting the blood sample with anti-inhibinantibodies that are specifically directed against the animal'sendogenous inhibin under conditions which allow the antibodies toselectively interact to any inhibin if present in the sample; removingany uninteracted antibodies from any interacted antibodies; anddetermining the quantity of antibodies that are interacted.

One of ordinary skill in the art will understand that the immunoassaytechniques that can be used in the above method are well known in theart. Therefore, any immunoassay technique, label, and visualizationmethod known in the art can be used in the above method, including ELISAand radioimmunoassay (RIA). A preferred immunoassay is ELISA ("enzymelinked immunosorbent assay"), and a preferred label is horseradishperoxidase. Another preferred label is a colored latex bead. The coloredlatex bead can be any color desired for visualization purposes.Preferably, the latex bead is yellow, red, blue, or green. The coloredlatex bead can be hollow or solid, but it preferably is hollow tominimize its weight. The size of the latex bead varies according to itsintended use in immunoassays. One of ordinary skill in the art would beable to ascertain by routine testing the largest bead size that isvisible yet does not interfere stearically with the immunoassayreactions. Preferably, the latex bead is less than 0.5μ in diameter, andmost preferably it is less than 0.2μ in diameter.

For example, circulating inhibin concentrations in the blood of a birdcan be determined using standard sandwich ELISA techniques. First, bindanti-inhibin antibodies which are directed against a portion of inhibin,or a fragment thereof, to the wells of a microtiter plate. After washingand blocking the plate, then add a quantity of blood plasma that wasobtained from the bird to be tested. After allowing any inhibin in thesample, if present, to selectively interact with the immobilizedanti-inhibin antibody, the sample is washed from the well of the plate.Next, add labeled anti-inhibin antibodies to the well that are directedagainst a different portion of the inhibin, or a fragment thereof, thanthe antibody immobilized in the well. The antibody can be labeled withany label known in the art, such as horseradish peroxidase. Afterallowing the labeled anti-inhibin antibody to selectively interact withany immobilized inhibin, any uninteracted labeled anti-inhibinantibodies are removed by washing. The amount of inhibin present in theplasma sample is determined by using the appropriate visualization meansfor the label used in the ELISA to quantify the amount of immobilizedlabeled anti-inhibin antibody in the well. Standard positive andnegative controls are to be run simultaneously in neighboring platewells.

It is to be understood that the reproductive potential of any animalwhich produces inhibin can be determined by the above method. The methodof the present invention can be used to determine the amount of inhibinproduced by a female animal of any species that produces inhibin. Theanimal can be a bird, mammal, fish, or reptile, among others. Morespecifically, the mammal includes, but is not limited to, a cow, human,horse, cat, dog, sheep, mink, fox, otter, ferret, raccoon, and pig. Thebird includes, but is not limited to, an ostrich, emu, and chicken. Apreferred animal is a bird. A more preferred animal is a ratite. Anespecially preferred animal is an ostrich. Another preferred animal isan emu. Yet another preferred animal is a chicken.

Animals having high levels of inhibin have low reproductive potential,and depending upon the species involved and whether they are raised foragricultural purposes, such a low egg producing animal may be taken toslaughter instead of being kept for breeding purposes. In contrast,those agriculturally raised animals having lower amounts of inhibin arehigher egg producers and generally are used for breeding purposes, andare not taken to slaughter.

The inhibin level in an animal varies according to its age, species, andthe time of year relative to the breeding season (if any). Therefore,the determination of the egg laying potential of an animal is relativeto the these factors, and a measure of an animal's inhibin levels aremost valuable when compared to the average inhibin levels of the samespecies of animal, of approximately the same age, at the same time ofyear if the animal has a breeding season.

As the amount of inhibin produced by a bird varies according to theabove factors, relative amounts of inhibin are established below fordifferent age categories of birds. Table 1 illustrates the variance ofratites such as emus and ostriches in their inhibin production dependentupon whether they are poor or good egg producers, and dependent upon theage of the ratite.

                  TABLE 1                                                         ______________________________________                                        Age            Inhibin     Reproductive                                       (Months)       Level       Potential                                          ______________________________________                                        6-12           >5          Poor                                               6-12           2-5         Moderate                                           6-12           0-1.5       Good                                               24+            >7          Poor                                               24+            3-7         Moderate                                           24+            0-2.5       Good                                               ______________________________________                                    

In the above table, the inhibin level is relative to a standard poolvalue of 1 for female ratites averaging 50 to 60 eggs per breedingseason, which is good, and a value of 7 for a pool of functionallynon-producing female ratites which produce less than 5 eggs per season,which is poor.

Yet another aspect of the present invention is a method of producinganimal antibodies directed against a class of another animal'santibodies, such as IgG. Briefly described, the method of producingantibodies in an animal which are directed against another animal's IgG,comprises the steps of: administering an effective amount of a class ofa first animal's antibodies, such as those produced by the abovedescribed method, to a second animal such that an immunological responseoccurs in the second animal against the first animal's antibodies;withdrawing a blood sample from the second animal; and isolating thesecond animal's antibodies from the serum of the blood sample asdescribed above. Preferably, the second animal is a different speciesthan the first animal.

The present invention is also directed to a rapid, simple, reliable,cost-effective method for determining whether an animal hasimmunologically responded to a challenge with an inhibin composition.This method utilizes a second animal's antibodies directed against aclass of antibodies from a first animal as described above. Brieflydescribed, the method comprises binding inhibin or the heterologousprotein of the present invention, to a solid phase, and contacting theimmobilized inhibin with a sample of blood from the animal to be tested.The sample is contacted with the immobilized inhibin under conditionswherein the inhibin will selectively interact with any anti-inhibinantibodies in the sample. After removing uninteracted antibodies fromthe sample and washing, a quantity of labeled antibodies from a secondanimal which are directed against a class of the first animal'santibodies are added. The labeled antibodies which are directed againstthe animal antibodies will then selectively interact with the antibodieswhich are bound to the immobilized inhibin. After removal of theuninteracted labeled antibodies, the presence or quantity of interactedlabeled antibodies is determined by visualizing the label. Thus, themethod detects the presence of antibodies directed against inhibin inthe animal, and therefore determines if the animal has immunologicallyresponded to the administration of a composition comprising inhibin.

It is to be understood that the method of determining if an animal hasimmunologically responded to the administration of a compositioncomprising inhibin can be performed on a female animal of any species.The animal can be a bird, mammal, fish, or reptile, among others. Morespecifically, the mammal includes, but is not limited to, a cow, human,horse, cat, dog, sheep, mink, fox, otter, ferret, raccoon, and pig. Thebird includes, but is not limited to, an ostrich, emu, a rhea, andchicken. A preferred animal is a bird. A more preferred animal is aratite. An especially preferred animal is an ostrich. Another preferredanimal is an emu. Yet another preferred animal is a rhea.

One of ordinary skill in the art will understand that the immunoassaytechniques that can be used in the above method are well known in theart. Therefore, any immunoassay technique, label, and visualizationmethod can be used in the above method. A preferred immunoassay isELISA, and a preferred label is horseradish peroxidase. Anotherpreferred label is a colored latex bead. The colored latex bead can beany color desired for visualization purposes. Preferably, the latex beadis yellow, red, blue, or green. The colored latex bead can be hollow orsolid, but it preferably is hollow to minimize its weight. The size ofthe latex bead varies according to its intended use in immunoassays. Oneof ordinary skill in the art would be able to ascertain the largest beadsize that is visible yet does not interfere stearically with theimmunoassay reactions. Preferably, the latex bead is less than 0.5μ indiameter, and most preferably it is less than 0.2μ in diameter.

Another embodiment of the present invention is directed to the abovemethod of determining if an animal has immunologically responded to theadministration of a composition comprising inhibin wherein theimmunoassay method is modified as follows. Briefly described, the methodcomprises obtaining a sample of blood from an animal, and contacting itwith labeled animal inhibin, or a fragment thereof. The sample iscontacted with the labeled animal inhibin under conditions wherein theanimal inhibin will selectively interact with any anti-inhibinantibodies in the sample. After removing uninteracted labeled inhibinfrom the sample, the presence or quantity of interacted labeled inhibinis determined by visualizing the label. The inhibin used in this methodis selected from, but is not limited to: the fused heterologous inhibinprotein of the present invention; endogenous inhibin, or fragmentsthereof; and exogenous inhibin, or fragments thereof. Preferably, thelabeled inhibin is endogenous inhibin.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1 Producing a Fused Gene Product Comprising a Gene Encoded forExpressing Chicken Inhibin, and a Gene Encoded for Expressing MaltoseBinding Protein.

The following is a method for producing a fused gene product comprisinga gene (cINA₅₂₁) encoded for expressing a fragment of alpha-subunitchicken inhibin (SEQ ID NO:2), and a gene encoded for expressing maltosebinding protein. The fused gene product of the present invention is madefrom the pMAL™-c vector kit, from New England Biolabs, Beverly, Mass.

The pMALTM vectors provide a method for producing a protein expressedfrom a gene cloned in a reading frame. The cloned gene is inserteddownstream from a gene, which encodes maltose-binding protein ("MBP"),and results in the expression of an MBP fusion protein ("MBP-cINA₅₂₁ ").The method yields high-level expression of the cloned sequences, and aone-step purification for the fusion protein, MBP-cINA₅₂₁, using MBP'saffinity for maltose.

The following is a Method of Ligating the Inhibin Gene, cINA₅₂₁, into apMAL™-c Vector:

1. Digest 0.5 μg pMAL™-c plasmid DNA in 20 μl with the restrictionendonuclease Pst1.

2. Digest 5 μg of cINA6 plasmid DNA which contains the chicken inhibingene with the same enzyme, Pst1.

3. Check for complete digestion by running 4 μl of the pMALT™ reactionand 4 μl of the cINA6 reaction on a 0.8% agarose gel. Then runpreparative agarose gel and purify the Pst1 cINA₅₂₁ fragment byprep-A-Gene purification kit.

4. Add 0.05 Units of calf intestinal alkaline phosphatase (NEB #290) tothe vector DNA digestion. Incubate at 37° C. for 1 hour.

5. Add an equal volume of a 1:1 phenol/chloroform mixture to the vectorrestriction digest, mix, and after centrifuging, remove the aqueous(top) phase and place in a fresh tube. Repeat with chloroform alone.

6. Add 10 μg glycogen or tRNA to the vector digest as carrier, add1/10th volume 3 M sodium acetate, mix, then add two volumes ethanol.Incubate at -20° C. for 30 minutes.

7. Microcentrifuge for 15 minutes. Pour off the supernatant, rinse thepellet with 70% ethanol, and allow to dry.

8. Resuspend each sample in 20 μl of water.

9. Mix: 0.2 μg vector digest; 0.5 μg insert digest; add water, up to 18μl, then add 2 μl of 10× ligase buffer; 0.5 μg NEB T4 ligase (#202;˜200units); and incubate at 16° C. for 2 hours to overnight.

10. Heat at 65° C. for 5 minutes; cool on ice.

11. Mix 5 μl of ligation mixture with 100 μl competent DH5α(or anylacZα-complementing strain) and incubate on ice for 15-30 minutes. Heatto 42° C. for 2 minutes.

12. Add 1 ml LB and incubate at 37° C. for 60 minutes. Spread on an LBplate containing 100 μg/ml ampicillin. Incubate overnight at 37° C. Pickcolonies with a sterile toothpick onto a master LB amp plate and an LBamp plate containing 80 μg/ml Xgal and 0.1 mM IPTG. Incubate at 37° C.for 8 to 16 hours. Score the Lac phenotype on the Xgal plate and recoverthe "white" clones from the master. 13. Screen for the presence ofinserts in one or both of the following ways:

A. Prepare miniprep DNA. Digest with an appropriate restrictionendonuclease to determine the presence and orientation of the insert.

B.

i) Grow a 5 ml culture in LB amp broth to about 2×10⁸ /ml.

ii) Take a 1 ml sample. Microcentrifuge for 2 minutes, discard thesupernatant and resuspend the cells in 50 μl protein gel SDS-PAGE samplebuffer.

iii) Add IPTG to the remaining culture to 0.3 mM, for example 15 μl of a0.1 M stock solution. Incubate at 37° C. with good aeration for 2 hours.

iv) Take a 0.5 ml sample. Microcentrifuge for 2 minutes, discard thesupernatant and resuspend the cells in 100 μl SDS-PAGE sample buffer.

v) Boil the samples 5 minutes. Electrophorese 15 μl of each sample on a10% SDS-PAGE gel along with a set of protein MW standards and 15 μl ofthe supplied MBP in SDS-PAGE sample buffer. Stain the gel with Coomassiebrilliant blue. An induced band is easily visible at a positioncorresponding to the molecular weight of the fusion protein. Themolecular weight of the MBP alone is 42,000 Daltons.

EXAMPLE 2 Producing a Fused Heterologous Protein, "MBP-cINA₅₂₁ ",Comprising Chicken Inhibin and Maltose Binding Protein.

The following is a method of producing a fused heterologous proteincomprising chicken inhibin and maltose binding protein, "MBP-cINA₅₂₁ ".The fused gene product of Example 1 expresses the fused heterologousmaltose binding protein-inhibin protein, "MBP-cINA₅₂₁ ", as follows:

1. Inoculate 80 ml rich broth+glucose and ampicillin (see Media andSolutions below) with 0.8 ml of an overnight culture of cells containingthe fusion plasmid of Example 1.

2. Grow at 37° C. with good aeration to 2×10⁸ cells/ml (A₆₀₀ of ˜0.5).Take a sample of 1 ml and microcentrifuge for 2 minutes (uninducedcells). Discard supernatant and resuspend the cells in 50 μl SDS-PAGEsample buffer. Vortex and place on ice.

3. Add IPTG (isopropylthiogalactoside) to the remaining culture to givea final concentration of 0.3 mM, e.g. 0.24 ml of a 0.1 M stock in H₂ O(see Media and Solutions). Continue incubation at 37° C. for 2 hours.Take a 0.5 ml sample and microcentrifuge for two minutes (inducedcells). Discard supernatant and resuspend the cells in 100 μl SDS-PAGE:sample buffer. Vortex to resuspend cells and place on ice.

4. Divide the culture into two aliquots. Harvest the cells bycentrifugation at 4000×g for 10 min. Discard the supernatant andresuspend one pellet (sample A) in 5 ml of lysis buffer (see Media andSolutions). Resuspend the other pellet (sample B) in 10 ml 30 mMTris-Cl, 20% sucrose, pH 8.0 (8 ml for each 0.1 g cells wet weight).

5. Freeze samples in a dry ice-ethanol bath (or overnight at -28° C.).Thaw in cold water (20° C. is more effective than 70° C., but takeslonger).

6. Sonicate, monitor cell breakage, by measuring the release of proteinusing the Bradford assay or the release of nucleic acid at A₂₆₀° untilit reaches a maximum. Add 0.6 ml 5M NaCl. 7. Centrifuge at 9,000×g for20 minutes. Decant the supernatant (crude extract 1) and save on ice.Resuspend the pellet in 5 ml lysis buffer. This is a suspension of theinsoluble matter (crude extract 2).

Column Purification of Heterologous Fused Maltose BindingProtein-Inhibin Protein, "MBP-cINAS521", as Produced Above, is asFollows:

1. Swell amylose resin (1.5 g) for 30 min. in 50 ml column buffer (seeMedia and Solutions) in a 250 ml filter flask. De-gas with an aspirator.Pour in a 2.5×10 cm column. Wash the column with 3 column volumes of thesame buffer +0.25% Tween 20. The amount of resin needed depends on theamount of fusion protein produced. The resin binds about 3 mg/ml bedvolume, so a column of about 15 ml should be sufficient for a yield ofup to 45 mg fusion protein/liter culture. A 50 ml syringe plugged withsilanized glass wool can be substituted for the 2.5 cm column. Thecolumn height to diameter ratio should be less than or equal to 4.

2. Dilute the crude extract 1:5 with column buffer +0.25% Tween 20. Loadthe diluted crude extract at a flow rate of 10×(diameter of column incm)² ! ml/hr. This is about 1 ml/min. for a 2.5 cm column. The dilutionof the crude extract is aimed at reducing the protein concentration toabout 2.5 mg/ml. If the crude extract is less concentrated, do notdilute it as much. A good rule of thumb is that 1 g wet weight of cellsgives about 120 mg protein.

3. Wash with 2 column volumes column buffer +0.25% Tween 20.

4. Wash with 3 column volumes column buffer without Tween 20.

5. Elute the fusion protein, "MBP-cINA₅₂₁ ", with column buffer +10 mMmaltose +0.1% SDS (optional 10 mM β-mercaptoethanol, 1 mM EGTA). Collect10-20 3 ml fractions. Assay the fractions for protein, e.g., by theBradford assay or A₂₆₀° ; the fractions containing the fusion proteinhave easily detectable protein. The fusion protein elutes soon after thevoid volume of the column.

Media and Solutions

Rich medium +glucose and ampicillin=per liter: 10 g tryplone, 5 g yeastextract, 5 g NaCl, 2 g glucose. Autoclave; add sterile ampicillin to 100μg/ml.

0.1 M IPTG Stock=1.41 g IPTG (isopropyl-β-o-thiogalactoside); add H₂ Oto 50 ml. Filter, and sterilize.

0.5 M sodium phosphate buffer, pH 7.2 (stock)=

(A) 69.0 g NaH₂ PO₄ H₂ O to 1 liter with H₂ O.

(B) 70.9 g Na₂ HPO₄ to 1 liter with H₂ O.

Mix 117 ml (A) with 383 ml (B). The pH of this stock should be ˜7.2.Diluted to 10 mM in column buffer, the pH should be 7.0.

    ______________________________________                                        Per Liter             Final Concentration                                     ______________________________________                                        Lysis Buffer                                                                  20 ml 0.5 M Na.sub.2 HPO.sub.4                                                                      10 mM phosphate                                         1.75 g NaCl           30 mM NaCl                                              10 ml 25% Tween 20    0.25% Tween j20                                         0.7 ml β-mercaploethanol                                                                       10 mM β-ME                                         ("β-ME") (optional)                                                      20 ml 0.5 M BDTA (pH 8)                                                                             10 mM EDTA                                              10 ml 1 M EGTA (pH 7) 10 mM EGTA                                              Adjust to pH 7.0 with HCL or                                                  NaOH                                                                          Column Buffer                                                                 20 ml 0.5 M sodium phosphate,                                                                       10 mM phosphate                                         pH 7.2                                                                        29.2 g NaCl           0.5 M NaCl                                              1 ml 1 M sodium azide 1 mM azide                                              0.7 mM β-ME (optional)                                                                         10 mM β-ME                                         1 ml 1 M EGTA (pH 7)  1 mM EGTA                                               (optional)                                                                    Adjust to pH 7.0 if necessary.                                                Low Salt Column Buffer                                                        20 ml 0.5 M sodium phosphate,                                                                       10 mM phosphate                                         pH 7.2                                                                        1.75 g NaCl           30 mM NaCl                                              1 ml 1 M sodium azide 1 mM azide                                              0.7 ml β-mercaptoethanol                                                                       10 mM β-ME                                         (optional)                                                                    1 ml 1 M EGTA (pH 7)  1 mM EGTA                                               (optional)                                                                    Adjust to pH 7.0 if necessary.                                                ______________________________________                                    

The purity of the fused chicken inhibin-MBP heterologous protein,"MBP-cINA₅₂₁ ", after passing through the column is illustrated in FIG.1, columns "E". The column marked "F" is the eluent from the column whenno heterologous protein has been loaded on the column (the negativecontrol). The columns marked "B" represent the plasmid pMAL™-c vectorstandards. The columns marked "C" are molecular weight standards. Thecolumns marked "D" are the actual pMAL™-c vector used in the preparationof the fused chicken-inhibin-MBP heterologous protein, "MBP-cINA₅₂₁ ",prior to the insertion of the inhibin gene as described in Example 2.The above proteins were electrophoresed on a SDS-PAGE gel in SDS-PAGEsample buffer, and stained with Coomassie brilliant blue stain.

EXAMPLE 3 Immunizing an Ostrich Against Inhibin.

The following is a method for immunizing an ostrich against inhibin. Aprimary immunization is administered to the ostrich approximately sixmonths prior to the bird's first breeding season, and then boosterimmunizations are administered at one month intervals for six months.Accordingly, it is preferable to administer the primary immunization toan ostrich when it is between approximately 18 and 24 months old. Theprimary immunization comprises between approximately 1.5 to 3.0 mg of afused heterologous protein comprising chicken inhibin (a fragment of thealpha subunit) and maltose binding protein produced by the methodsdescribed in Examples 1 and 2. The booster immunizations comprisebetween approximately 0.75 to 1.5 mg of the fused heterologous protein.The fused heterologous protein is emulsified in Freund's CompleteAdjuvant (Sigma Chemical Co., St. Louis, Mo.) in the primaryimmunization, and the fused heterologous protein is emulsified inFreund's Incomplete Adjuvant (Sigma) in the booster immunizations. Thefused heterologous protein composition is injected subcutaneously atthree sites along the upper thigh region of the ostrich.

EXAMPLE 4 Producing Ostrich Antibodies Selectively Directed AgainstInhibin.

The following is a method of producing ostrich antibodies ("ostrichanti-chicken inhibin antibodies") which are selectively directed againsta heterologous protein of the present invention comprising chickeninhibin and maltose binding protein. More particularly, the ostrichantibodies are IgG antibodies. The heterologous protein used is a fusedprotein comprising chicken inhibin and maltose binding protein producedby the methods described in Examples 1 and 2. To produce the antibodies,immunize an ostrich with chicken inhibin-MBP heterologous proteinaccording the method described in Example 3. The amount administered tothe ostrich must be sufficient to ellicit an immunological response inthe ostrich against the heterologous protein. Withdraw approximately 5ml of blood from the ostrich, and then isolate antibodies directedagainst inhibin from the remainder of the blood sample. Any separationmethod known in the art can be used to isolate the antibodies.Preferably, standard ELISA techniques are employed in conjunction withaffinity and HPLC columns.

EXAMPLE 5 Producing Goat Antibodies Selectively Directed Against OstrichAntibodies.

The following is a method of producing goat antibodies which areselectively directed against a class of ostrich antibodies, includingthe antibodies produced in Example 4, more particularly IgG ostrichantibodies. In this method a goat is immunized with 0.5 to 3.0 mgostrich IgG such that an immunological response occurs in the goatagainst the ostrich IgG. The ostrich IgG are obtained from a pool ofsera from different ostriches. Blood is obtained from the goat, and goatanti-ostrich IgG is then isolated from the sample using standardtechniques that are well known in the art.

The pool of sera is purified using standard methods that involveprecipitation using 50% ammonium sulfate, and subsequent fractionationusing a protein-A sepharose column. Preferably, the IgG precipitation isconducted as follows. Twelve milliliters of sera containing IgG isdiluted 1:1 with 50 mM-Tris (pH 8.0). Next, 24 ml of saturated ammoniumsulphate is added slowly with stirring (all at 4° C.), and the mixtureis stirred for approximately 2 hours. Centrifuge the mixture at 10,000rpm for 10 minutes to collect the precipitate. Resuspend the precipitatein 50 mM-Tris/Saline (to 12 ml), and dialyse overnight versus 2L of50mM-Tris at 4° C., the final volume being 20 ml.

Preferably, the subsequent fractionation using a protein-A sepharosecolumn is as follows. The column contains protein-A sepharose CL-4B(Pharmacia Biotech, Inc., Piscataway, NJ). The column is loaded withapproximately 5 ml of ammonium sulfate/serum precipitate. The sample isallowed to bind to the protein A-sepharose for approximately 30 minutes.Next, the column is washed with 0.1 M phosphate buffer (pH 7.5), and theabsorbed IgG is eluted with 0.1 M Glycine (pH 2.8). Finally, the elutedfractions are neutralized by adding a few drops of lM Tris-HCl (pH 9).The quality of the purified ostrich IgG is tested by visualization afterSDS-polyacrylamide gel electrophoresis before it is administered to agoat.

The method of immunization and the adjuvants used are not critical tothe invention, thus any method known in the art can be used, and anyadjuvant system known in the art can be used. Preferably, the purifiedostrich IgG will be injected into a goat subcutaneously. It is alsopreferable to administer boost injections at four week intervals usingFreund's incomplete adjuvant. Preferably, the purified IgG will beadministered with Freund's Complete Adjuvant or Hunter's Titermax (SigmaChemical Co., St. Louis, Mo.). A goat develops a satisfactory immuneresponse after three to four injections.

Next, 5-10 ml of blood is withdrawn from the goat, and the goatantibodies directed against ostrich IgG are isolated from the bloodsample. Any method known in the art can be used to separate the goatantibodies from the blood sample. A preferred method for separating thegoat antibodies from the sample is by passing the blood sample throughan ostrich IgG column. The goat antibodies are then collected from thecolumn by washing the column with glycine buffer-pH 8.0.

EXAMPLE 6 Monitoring the Immunological Response of an Ostrich after itsVaccination with a Fused Heterologous Protein.

The following is a method of monitoring the immunological response of anostrich after it has been vaccinated with a fused heterologous proteincomprised of chicken inhibin and maltose binding protein produced by themethod of Examples 1 and 2, wherein the immunological response ismonitored using the goat antibodies produced in Example 5. To determinewhether an ostrich has immunologically reacted to the fused inhibin-MBPheterologous protein, first bind the heterologous protein to a solidphase. Next, withdraw 5-10 ml of blood from an ostrich that has beenimmunized with the heterologous protein, and isolate the serum from theblood. Contact the immobilized heterologous protein with the serum underconditions wherein the heterologous protein will selectively interactwith any anti-inhibin antibodies in the serum. After washing, add thegoat antibodies produced in Example 5 which have been labeled with HRP.The labeled goat antibodies will then selectively interact with theostrich antibodies which are bound to the immobilized heterologousprotein. After the removal of the uninteracted labeled antibodies, thepresence or quantity of interacted HRP labeled goat antibodies isdetermined by visualizing the label. Preferably, the label is visualizedby adding Nitro Blue Tetrazolium ("NBT"), a substrate to HRP.

One of ordinary skill in the art will understand that the immunoassaytechniques that are used in the above method are well known in the art.Therefore, any immunoassay technique, label, and visualization methodcan be used in the above method. A preferable immunoassay is ELISA, anda preferable label is horseradish peroxidase. Another preferable labelis a colored latex bead.

EXAMPLE 7 Determining the Reproductive Potential of an Ostrich.

The following is a method for determining the reproductive potential ofan ostrich by quantifying the amount of inhibin in the ostrich's blood.Circulating inhibin concentrations in the blood of an ostrich can bedetermined using radioimmunoassay (RIA) or standard sandwich ELISAtechniques. First, bind the anti-inhibin antibodies produced in Example4, to the wells of a microtiter plate. After washing and blocking theplate, then add a quantity of blood plasma or serum that was obtainedfrom the ostrich to a well of the microtiter plate. After allowing anyinhibin in the sample, if present, to selectively interact with theimmobilized anti-inhibin antibodies, the sample is washed from the wellof the plate. Next, add a different anti-inhibin antibody to the wellthan that produced in Example 4, which is conjugated with horseradishperoxidase. The HRP conjugated anti-inhibin antibody differs from theimmobilized anti-inhibin body in that they are selectively directedagainst different portions of inhibin. After allowing the labeledanti-inhibin antibody to selectively interact with any immobilizedinhibin, any uninteracted labeled anti-inhibin antibodies are removed bywashing. The amount of inhibin present in the plasma sample isdetermined by adding NBT to the well and visualizing the amount ofimmobilized labeled anti-inhibin antibody in the well. Standard positiveand negative controls are run simultaneously in neighboring plate wells.Many immunoassay techniques, labels and visualization methods are wellknown in the art. Accordingly, any immunoassay method, label, andvisualization technique can be used in the present invention.

EXAMPLE 8 Enhancing Egg Production in Quail.

As stated above, the chicken inhibin α-subunit cDNA clone (cINA6)inserted into the EcoR 1 site of Bluescript was obtained as a gift of P.A. Johnson (Cornell University). A DNA fragment ("cINA₅₂₁ ") was excisedfrom the cINA6 clone using Pst 1 digestion. The cINA₅₂₁ DNA fragmentencompassed most of the mature chicken inhibin α-subunit. This fragment(cINA₅₂₁) was cloned in plasmid p-MAL™-c in frame with the maltosebinding protein ("MBP") and a fusion protein of appropriate size (LaneE; FIG. 1) was detected after IPTG (isopropyl β-D-thiogalactopyranoside)induction and SDS-PAGE. The resulting protein conjugate ("MBP-cINA₅₂₁ ")was used as an antigen to immunize pre-pubescent, female Japanese quail(Coturnix coturnix japonica) against circulating inhibin levels asdescribed below.

Hatchling quail were brooded in a Model 2S-D Petersime brooder batterymodified for quail. Initial brooding temperature was approximately 37.8°C. with a weekly decline of approximately 2.8° C. until ambienttemperature was achieved. During the growing period (ie., untilapproximately 6 wks-of-age), a quail starter ration (28% CP, 2,800 kcalME/kg of feed) and water were provided for ad libitum consumption, andcontinuous dim light (22 1×) with a 14 h light (280 to 300 1×):10 hrdark override was used. At 25 days-of-age, 50 quail were randomly andequally assigned to one of two injection groups (25 birds per group) asfollows: (1) MBP-cINA₅₂₁ in Freund's adjuvant ("MBP-cINA₅₂₁ /FRN"), or(2) Freund's (adjuvant control; "FRN"). Birds immunized against inhibin(Group 1) were given approximately 0.75 mg MBP-cINA₅₂₁ per bird in theappropriate control vehicle. Equivalent vehicular injection volumes (0.2mL) of FRN were administered to Group 2. All injections were givensubcutaneously using tuberculin syringes fitted with 25 ga needles.Following the initial injections, quail were wingbanded to identify themby treatment before housing (individually) in laying cages. Weeklybooster inhibin immunizations of approximately 0.375 mg MBP-cINA₅₂₁ perbird, or appropriate control challenges, were subsequently administeredfor five consecutive weeks (ie., at 32, 39, 46, 53, and 60 days-of-age)and then every 35 days thereafter for three additional challenges (i.e.,at 95, 130, and 165 days-of-age). Beginning at 6 weeks-of-age, a quailbreeder ration (21% CP, 2,750 kcal ME/kg of feed) and water wereprovided for ad libitum consumption.

Beginning at 41 days-of age (considered Day 1 of the egg lay cycle),daily hen-day egg production ("HDEP") and mortality ("MORT") measureswere recorded for 20 consecutive weeks. In addition, average age atfirst egg lay ("FIRST") and age at which hens reached 50% egg production("FIFTY"), or maximum egg lay as defined above, were calculated for eachof the treatment groups.

Hen-day egg production data were subjected to an analyses of variance("ANOVA") of which incorporated a completely randomized design with asplit-plot arrangement of treatments. The main plot consisted of the twoinjection treatments (MBP-cINA₅₂₁ /FRN, or FRN) and the 20 layingperiods of 7 days each comprised the split.

Inhibin immunoneutralization clearly accelerated puberty in the quailhens. The average age of FIRST egg lay was decreased (P<0.0088) bynearly six days in inhibin-treated hens (Table 2). Likewise, the age toFIFTY egg production was markedly reduced (12 days; P<0.01) ininhibin-treated hens (Table 3).

A positive effect of inhibin treatment on hen day egg production (HDEP)was also extant, most notably at the beginning and at the end of thelaying cycle (FIG. 2). For example, significantly greater (P<0.05) meanHDEP rates were observed in hens treated with MBP-cINA₅₂₁ /FRN whencompared to the FRN controls during Weeks 1 (16.5 vs 2.6%), 2 (50.0 vs28.6%), and 4 (96.6 vs 79.7%) and again during Weeks 15 (98.8 vs 86.9%),16 (96.9 vs 86.3%), 18 (85.7 vs 66.1%), and 20 (96.8% vs 73.8%). TotalHDEP rate (inclusive of all 20 weeks of lay) for inhibin-treated henswas 83.5% as compared to 75.4% for the controls.

Besides accelerating puberty, prolonging egg lay, and enhancing theoverall intensity of lay, inhibin-treatment decreased the time needed toreach peak egg lay by approximately 3 weeks (FIG. 2; Compare MBP-cINA₅₂₁/FRN=96.6% HDEP by Week 4 vs FRN=96.6% HDEP by Week 7). Althoughdifferences in peak HDEP values were not statistically evaluated, thetreatment differences in mean age at which hens reached 50% HDEP levels(FIFTY) reflect peak performance.

Mortality was not a factor in this study as only eight birds have died(three controls, five treated). Such MORT (16%) would be within expectedlimits for quail that have reached 180 days-of-age.

                  TABLE 2                                                         ______________________________________                                        Effect of inhibin immunoneutralization on mean (±SE)                       age at first egg lay in Japanese quail                                                            Age at first egg lay                                      Treatment           (days)                                                    ______________________________________                                        FRN.sup.1           56.15 ± 1.82.sup.a                                     MBP-cINA.sub.521 /FRN.sup.2                                                                       50.38 ± 1.08.sup.b                                     ______________________________________                                         .sup.1 Freund's adjuvant Control.                                             .sup.2 MBPcINA.sub.521 /FRN = Maltose Binding Proteinchicken                  α.sub.515inhibin fusion protein in Freund's adjuvant.                   .sup.a,b (P < .0088).                                                    

                  TABLE 3                                                         ______________________________________                                        Effect of inhibin immunoneutralization on mean (±SE)                       age at 50% egg production in Japanese quail                                                        Age at 50% egg                                           Treatment            production (days)                                        ______________________________________                                        FRN.sup.1            73.04 ± 3.78.sup.a                                    MBP-cINA.sub.521 /FRN.sup.2                                                                        61.00 ± 2.70.sup.b                                    ______________________________________                                         .sup.1 Freund's adjuvant control.                                             .sup.2 MBPcINA.sub.521 /FRN = Maltose Binding Proteinchicken                  α.sub.515inhibin fusion protein in Freund's adjuvant.                   .sup.a,b (P < .01).                                                      

Incidences of shelless (unshelled) and thin-shelled eggs occurred atgreater frequencies in control birds, particularly 25 during the latterstages of the laying cycle, than in inhibin-immunized birds. Thissuggests that greater numbers of defective eggs (i.e., eggs that wereeither unfit for consumption or likely to break before consumption, orunsettable as hatching eggs) were associated with the control treatment.

EXAMPLE 9 Enhancing Production Performance in Ostriches.

The protein conjugate (MBP-cINA₅₂₁) is used as an antigen to immunizeprepubescent, female ostriches against circulating inhibin levels, andto therefore accelerate the onset of egg lay in the treated ostriches.The method described in Example 8 is followed with the followingexceptions. The average age at puberty for untreated ostriches isbetween approximately 28 and 32 months. The following is the treatmentschedule for ostriches having an approximate body weight range of 150 to300 pounds: primary (first) injection of 5.0 mg of the heterologousprotein of the present invention on its 26th month of age; and boostersof 2.5 mg on the 27th, 28th, 30th, 32nd, 34th, and 36th month of age.

EXAMPLE 10 Enhancing Production Performance in Emu.

The protein conjugate (MBP-cINA₅₂₁) is used as an antigen to immunizeprepubescent, female emu against circulating inhibin levels, and totherefore accelerate the onset of egg lay in the treated emu. The methoddescribed in Example 8 is followed with the following exceptions. Theaverage age at puberty for untreated emu is approximately 20 months. Thefollowing is the treatment schedule for emu having an approximate bodyweight range of 50 to 90 pounds: primary (first) injection of 3.0 mg ofthe heterologous protein of the present invention on its 18th month ofage; and boosters of 1.5 mg on the 19th, 20th, 22nd, 24th, 26th, and30th month of age.

EXAMPLE 11 Enhancing Production Performance in Chickens.

The protein conjugate (MBP-cINA₅₂₁) is used as an antigen to immunizeprepubescent, female chickens against circulating inhibin levels, and totherefore accelerate the onset of egg lay in the treated ostriches. Themethod described in Example 8 is followed with the following exceptions.The average age at puberty for an untreated chicken is approximately 20weeks. The following is the treatment schedule for a chicken having anapproximate body weight range of 2.0 to 3.5 pounds: primary (first)injection of 1.5 mg of the heterologous protein of the present inventionon its 15th week of age; and boosters of 0.75 mg on the 17th, 20th,24th, 30th, 40th, and 50th week of age.

EXAMPLE 12 Enhancing Production Performance in Turkeys.

The protein conjugate (MBP-cINA₅₂₁) is used as an antigen to immunizeprepubescent, female turkeys against circulating inhibin levels, and totherefore accelerate the onset of egg lay in the treated turkeys. Themethod described in Example 8 is followed with the following exceptions.The average age at puberty for an untreated turkey is approximately 30weeks. The following is the treatment schedule for a turkey having anapproximate body weight range of 9.0 to 12 pounds: primary (first)injection of 2.0 mg of the heterologous protein of the present inventionon its 28th week of age; and boosters of 1.0 mg on the 29th, 30th, 34th,39th, 46th, and 54th week of age.

EXAMPLE 13 Enhancing Production Performance in Parrots.

The protein conjugate (MBP-cINA₅₂₁) is used as an antigen to immunizeprepubescent, female parrots against circulating inhibin levels, and totherefore accelerate the onset of egg lay in the treated parrots. Themethod described in Example 8 is followed with the following exceptions.The average age at puberty for an untreated parrot is approximately 30months. The following is the treatment schedule for a parrot having anapproximate body weight range of 0.5 to 1.25 pounds: primary (first)injection of 0.7 5 mg of the heterologous protein of the presentinvention on its 28th month of age; and boosters of 0.375 mg on the29th, 30th, 32nd, 34th, 36th, and 38th month of age.

It should be understood, of course, that the foregoing relates only topreferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 521 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..303                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTGCAGCGCCCATCGGAGGACGTGGCCGCCCACACCAACTGCCGCCGG48                            LeuGlnArgProSerGluAspValAlaAlaHisThrAsnCysArgArg                              151015                                                                        GCGTCCCTCAACATCTCTTTCGAGGAGCTGGGCTGGGACAATTGGATC96                            AlaSerLeuAsnIleSerPheGluGluLeuGlyTrpAspAsnTrpIle                              202530                                                                        GTGCACCCCAGCAGCTTCGTTTTCCACTACTGCCACGGGAACTGTGCC144                           ValHisProSerSerPheValPheHisTyrCysHisGlyAsnCysAla                              354045                                                                        GAAGGCCACGGGCTGAGCCACCGGCTGGGGGTGCAGCTGTGCTGCGCC192                           GluGlyHisGlyLeuSerHisArgLeuGlyValGlnLeuCysCysAla                              505560                                                                        GCGCTGCCCGGCACCATGCGCTCACTGCGTGTCCGCACCACCTCTGAT240                           AlaLeuProGlyThrMetArgSerLeuArgValArgThrThrSerAsp                              65707580                                                                      GGTGGCTACTCCTTCAAGTACGAGACGGTGCCCAACATCCTGGCGCAG288                           GlyGlyTyrSerPheLysTyrGluThrValProAsnIleLeuAlaGln                              859095                                                                        GACTGCACCTGTGTCTAGCAGCTGGCATGCACGGCCAGACCCGCGTGGATCTCCC343                    AspCysThrCysVal                                                               100                                                                           CGTTGCCTCTGGACTGCCCCAGTGCCAGATGATGAGCCCATCCCAGGGATGGAGGAGTCA403               CTCACACGGGCACTGCGCAGCCCGGAGCAGGGAGAGGGACCCAGGTGGAAGTTTTGGTGG463               TGCCACCCTCCCTTTGACTGCCAGGGTTTCATGGTTTCAGGTTGCGTGGGTGCTGCAG521                 (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuGlnArgProSerGluAspValAlaAlaHisThrAsnCysArgArg                              151015                                                                        AlaSerLeuAsnIleSerPheGluGluLeuGlyTrpAspAsnTrpIle                              202530                                                                        ValHisProSerSerPheValPheHisTyrCysHisGlyAsnCysAla                              354045                                                                        GluGlyHisGlyLeuSerHisArgLeuGlyValGlnLeuCysCysAla                              505560                                                                        AlaLeuProGlyThrMetArgSerLeuArgValArgThrThrSerAsp                              65707580                                                                      GlyGlyTyrSerPheLysTyrGluThrValProAsnIleLeuAlaGln                              859095                                                                        AspCysThrCysVal                                                               100                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTCAGCCTGCTGCAGCGCCC20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTGTCGACCGCGCGACGCCGAC22                                                      __________________________________________________________________________

What is claimed is:
 1. A fused heterologous protein which increases egglay, accelerates onset of egg lay, accelerates onset of maximum egg lay,increases lifetime total egg lay, increases egg production, increasesintensity of egg production, prolongs persistence of egg lay, improvesegg shell quality, accelerates onset of puberty, accelerates onset ofovulation, prolongs persistence of ovulation, increases spermproduction, accelerates onset of sperm production, accelerates onset ofmaximum sperm production, prolongs persistence of sperm production,improves sperm viability, increases testosterone production, increasesejaculate volume, or increases libido, when administered in an effectiveamount to a bird, the protein comprising avian alpha-subunit inhibinprotein, or an immunogenic fragment thereof, fused to a carrier protein.2. The fused heterologous protein of claim 1, wherein the avianalpha-subunit inhibin protein, or an immunogenic fragment thereof,comprises the sequence of SEQ ID NO:
 2. 3. The fused heterologousprotein of claim 1, wherein the carrier protein is maltose bindingprotein, bovine serum albumin, ovalbumin, flagellin, keyhole-limpethemocyanin, serum albumin, gamma globulin, thyroglobulin, or polymers ofamino acids.
 4. A method of producing a fused heterologous proteincomprising the steps of:(a) providing double strand cDNA which isencoded for avian alpha-subunit inhibin protein, or an immunogenicfragment thereof; (b) providing a vector which has coding informationfor the production of a carrier protein; (c) ligating the avianalpha-subunit inhibin cDNA into the vector; (d) inserting the ligatedvector into an expression system; and (e) expressing a fusedheterologous protein comprising avian alpha-subunit inhibin protein, oran immunogenic fragment thereof, and the carrier protein.
 5. The methodof claim 4, wherein the avian alpha-subunit inhibin cDNA comprises thesequence of SEQ ID NO:
 1. 6. The method of claim 4, wherein the avianalpha-subunit inhibin protein, or immunogenic fragment thereof, in theexpressed fused heterologous protein comprises the sequence of SEQ IDNO:
 2. 7. The method of claim 4, wherein the carrier protein is maltosebinding protein, bovine serum albumin, ovalbumin, flagellin,keyhole-limpet hemocyanin, thyroglobulin, serum albumin, gamma globulin,or polymers of amino acids.